Cytokine polypeptides and nucleic acids

ABSTRACT

New cytokine polypeptides, and nucleic acids encoding them, are provided. Compositions including these polypeptides and nucleic acids, recombinant cells comprising said polypeptides and nucleic acids, methods of making the polypeptides and nucleic acid, antibodies to the polypeptides, and methods of using the polypeptides and nucleic acids are provided. Integrated systems comprising the sequences of the nucleic acids or polypeptides are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of and claims thebenefit of and priority to U.S. Patent Application Ser. No. 60/169,035,filed Dec. 2, 1999, the disclosure of which is incorporated herein byreference in its entirety for all purposes.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), a portion of this patent documentcontains material which is subject to copyright protection. Thecopyright owner has no objection to the facsimile reproduction by anyoneof the patent document or the patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates to the generation of new cytokinepolypeptides and nucleic acids which encode them.

BACKGROUND OF THE INVENTION

T helper (T_(H)) cells, key regulators of the immune system, are dividedinto two subsets, T_(H)1 and T_(H)2, based upon their pattern ofcytokine synthesis (Paul and Seder (1994) Cell 76: 241-251). T_(H)1cells predominantly produce high levels of interleukin-2 (IL-2) andinterferon-gamma (IFN-γ). T_(H)1 cells also activate antigen-presentingcells (macrophages and dendritic cells) and enhance the cytotoxicactivity of CD8⁺ Cytotoxic T-lymphocyte (CTL) and Natural Killer (NK)cells. In contrast, T_(H)2 cells produce elevated levels of IL-4, IL-5and IL-13, and mediate allergic responses as a result of inducing IgEisotype switching and differentiation of B cells into IgE secretingcells (De Vries and Punnonen (1996) In Cytokine regulation of humoralimmunity: basic and clinical aspects. Eds. Snapper, C. M., John Wiley &Sons, Ltd., West Sussex, UK, p. 195-215).

It is desirable to modify the relative populations of T_(H)1 and T_(H)2cells in various circumstances. Modulators which up-regulateT_(H)1-mediated responses by, for example, directing the differentiationof naive T cells into T_(H)1 cells, inducing T_(H)1 cell proliferation,and increasing IFN-γ production and macrophage activation, are useful inpromoting cell-mediated immunity to infectious agents such as bacterial,protozoan, intracellular parasitic and viral infections. Modulatorswhich down-regulate T_(H)1-mediated responses are useful in situationswhere a decreased cell-mediated immune response in desired, for example,in treatment of autoimmune diseases such as multiple sclerosis.

The discovery of novel cytokine polypeptides which modulateT_(H)1-mediated responses, and nucleic acids encoding them, satisfies aneed in the art by providing new compositions useful in modifying hostimmune responses and in treating disease.

SUMMARY OF THE INVENTION

The invention provides modified cytokine polypeptides (also referred toherein as “modified p40 polypeptides” and as “modified p35polypeptides”), nucleic acids encoding the polypeptides andcomplementary nucleotide sequences thereof, fragments of saidpolypeptides and nucleic acids, antibodies to the polypeptides, and usestherefor, a computer or computer readable medium comprising a sequencerecord comprising one or more data sets containing character strings ofnew cytokine sequences, and automated systems for using the characterstrings.

In one aspect, the invention includes an isolated or recombinant nucleicacid encoding a modified cytokine polypeptide of the invention. Includedare polynucleotide sequences comprising a mature polypeptide codingregion of a sequence selected from SEQ ID NO:1 to SEQ ID NO:7, or SEQ IDNO:16 to SEQ ID NO:35, and complementary polynucleotide sequencesthereof. Polynucleotide sequences encoding a polypeptide comprising amature polypeptide region of an amino acid sequence selected from SEQ IDNO:8 to SEQ ID NO:14 or SEQ ID NO:26 to SEQ ID NO:35, and complementarypolynucleotide sequences thereof, are also a feature of the invention.Similarly, a polynucleotide sequence which hybridizes under at leasthighly stringent conditions over substantially the entire length of anyone of the preceding polynucleotide sequences is a feature of theinvention. A polynucleotide sequence which encodes a polypeptidecomprising an amino acid sequence having at least about 90% sequenceidentity to a mature polypeptide region of a sequence selected from SEQID NO:8 to SEQ ID NO:14 and SEQ ID NO:39, or to a mature polypeptideregion of a sequence selected from SEQ ID NO:26 to SEQ ID NO:35 and SEQID NO:40, is also a feature of the invention. In various embodiments,the polynucleotide sequence encodes a polypeptide comprising an aminoacid sequence having at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to a mature polypeptide region of a sequence selected from SEQID NO:8 to SEQ ID NO:14 and SEQ ID NO:39, or to a mature polypeptideregion of a sequence selected from SEQ ID NO:26 to SEQ ID NO:35 and SEQID NO:40. In addition, a polynucleotide sequence comprising a nucleotidefragment of any of the preceding polynucleotide sequences, whichnucleotide fragment encodes a polypeptide having T-cell proliferativeactivity and/or IFN-gamma induction activity in T-cells (e.g., humanT-cells) in the presence of a p35 polypeptide or a p40 polypeptide isalso a feature of the invention.

Any of the polynucleotides described above may optionally include aleader peptide coding region, which encodes a leader peptide region. Inone embodiment, the optional leader peptide coding region encodes aleader peptide region comprising an amino acid sequence at least about90% identical to the amino acid sequence set forth as the leader peptideregion of any one of SEQ ID NO:8 to SEQ ID NO:14, or SEQ ID NO:26 to SEQID NO:35. In various embodiments, the optional leader peptide codingregion encodes a leader peptide region comprising an amino acid sequenceat least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence set forth as the leader peptide region of any one ofSEQ ID NO:8 to SEQ ID NO:14, or SEQ ID NO:26 to SEQ ID NO:35. In anotherembodiment, the optional leader peptide coding region encodes a leaderpeptide region comprising an amino acid sequence set forth as the leaderpeptide region of any one of SEQ ID NO:8 to SEQ ID NO:14, or SEQ IDNO:26 to SEQ ID NO:35.

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p40 polypeptidecomprising an amino acid modification located at an amino acid positionequivalent to (i.e., an “equivalent position” to) that in the amino acidsequence of a naturally-occurring or wild-type p40 polypeptide (SEQ IDNO:15). The modification can include: (a) a substitution of thespecified amino acid for a different amino acid at one or moreequivalent position to that of SEQ ID NO:15 selected from Leu62, Ser71,Gln78, His99, Thr127, Arg130, Lys185, Glu186, Tyr187, Glu188, Ser190,Asp196, Met211, Val289, Ser305, Ser307, Arg309, and Gln311; (b) adeletion of one or more amino acid residues at equivalent positionArg181 to Asn184 inclusive, or a substitution, of the amino acidresidues at equivalent positions Arg181 to Asn184 inclusive, for theamino acid residues Ser-(Leu or Met)-(Glu or Asp)-His-Arg; (c) adeletion of one or more amino acid residues at equivalent positionsAsp287 and Arg288. The modified p40 polypeptide may optionally includetwo or more of modification (a), (b) or (c). The modified p40polypeptide sequence encoded by the nucleic acid of the invention may bea modified sequence of a naturally-occurring (i.e., wild-type) p40polypeptide sequence of a mammal (e.g., human, a non-human primate, aruminant, or a rodent), preferably a primate, more preferably human. Themodified p40 polypeptide sequence encoded by the nucleic acid of theinvention may be a modified sequence of a polypeptide selected from thegroup consisting of p40 polypeptides encoded by nucleic acids having theGenBank accession numbers: M65272, M65290, U19841, U19834, Y11129,U83184, Y07762, AF054607, U49100, AF091134, U57752, U10160, AF007576,AF004024, U11815, U08317, X97019, AF082494, AF133197, U16674, M86671,S82426, AF097507, and AF046211. A modified polypeptide of the inventionmay comprise at least one of the substitutions Leu62Ser; Ser71Thr;Gln78His; His99(Arg or Gln); Thr127(Ser or Ile); Arg130Lys; Lys185Glu;Glu186Tyr; Tyr187(Lys or Asn); Glu188Lys; Ser190(Arg or Thr); Asp196Gly;Met211Val; Val289(Ile or Leu); Ser305Lys; Ser307Arg; Arg309Gln; andGln311Arg. A modified polypeptide of the invention may also comprise atleast one of the substitutions Lys27Glu; Asp29Asn; Asp40Asn; Met45Thr;Thr49Ala; Glu67Gly, His91Arg; Glu95(Ala or Thr), Val96Ala; Glu122Lys;Asn125Ala; Asn135Asp; Arg139His; Thr147Ala; Thr153Lys; Ser155Thr;Ser163Thr; Gln166(Arg or His), Ala172Thr; Ala173Val; Thr174Leu;Ala177Glu; Glu178Asp; Arg179Leu; Val180Gly; Ala201Ser; Val212Leu;Asp213Glu; Val215Ile; Ser226Arg; Lys244Arg; Gln251His; Val254Ile;Ser255Asn; Glu257Gly; Thr264(Ala or Ile); Thr272Met; Cys274Gly;Val275Ile; Lys280Arg; Ser281Asn; Lys285Asp; Lys286Arg; Phe290Ser;Thr291(Met or Val); Lys293Gln; Thr297Lys; Ile299(Thr or Val); Arg301His;Asn303Asp; Ser318Phe; Glu321Asp; Pro326Ser; Cys327Leu; and Ser328(Gly orGln).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p40polypeptide, wherein the modified p40 polypeptide comprises an aminoacid sequence having at least about 90% amino acid sequence identity tothe amino acid sequence identified herein as the mature polypeptideregion (amino acid residue positions 23-328) of SEQ ID NO:39:IWEL-X₂₇-K-X₂₉-VYVVELDWYP-X₄₀-APGE-X₄₅-VVL-X₄₉-CDTPEEDGITWT-X₆₂-DQSS-X₆₇-VLG-X₇₁-GKTLTI-X₇₈-VKEFGDAGQYTC-X₉₁-KGG-X₉₅-X₉₆-LS-X₉₉-SLLLLHKKEDGIWSTDILKDQK-X₁₂₂-PK-X₁₂₅-K-X₁₂₇-FL-X₁₃₀-CEAK-X₁₃₅-YSG-X₁₃₉-FTCWWLT-X₁₄₇-ISTDL-X₁₅₃-F-X₁₅₅-VKSSRGS-X₁₆₃-DP-X₁₆₆-GVTCG-X₁₇₂-X₁₇₃-X₁₇₄-LS-X₁₇₇-X₁₇₈-X₁₇₉-X₁₈₀-X₁₈₁-X₁₈₂-X₁₈₃-X₁₈₄-X₁₈₅-X₁₈₆-X₁₈₇-X₁₈₈-Y-X₁₉₀-VECQE-X₁₉₆-SACP-X₂₀₁-AEESLPIEV-X₂₁₁-X₂₁₂-X₂₁₃-A-X₂₁₅-HKLKYENYTS-X₂₂₆-FFIRDIIKPDPPKNLQL-X₂₄₄-PLKNSR-X₂₅₁-VE-X₂₅₄-X₂₅₅-W-X₂₅₇-YPDTWS-X₂₆₄-PHSYFSLTF-X₂₇₄-X₂₇₅-QVQG-X₂₈₀-X₂₈₁-KRE-X₂₈₅-X₂₈₆-X₂₈₇-X₂₈₈-X₂₈₉-F-X₂₉₁-D-X₂₉₃-TSA-X₂₉₇-V-X₂₉₉-C-X₃₀₁-K-X₃₀₃-A-X₃₀₅-I-X₃₀₇-V-X₃₀₉-A-X₃₁₁-DRY-X₃₁₅-SS-X₃₁₈-WS-X₃₂₁-WASV-X₃₂₆-X₃₂₇-X₃₂₈,or a conservatively substituted variation thereof, where X₂₇ is K or E;X₂₉ is D or N; X₄₀ is D or N; X₄₅ is M or T; X₄₉ is T or A; X₆₂ is S;X₆₇ is E or G; X₇₁ is T; X₇₈ is H; X₉₁ is H or R; X₉₅ is E, A, K, or T,X₉₆ is V or A; X₉₉ is R or Q; X₁₂₂ is E or K; X₁₂₅ is N or A; X₁₂₇ is Sor I; X₁₃₀ is K; X₁₃₅ is N or D; X₁₃₉ is R or H; X₁₄₇ is T or A; X₁₅₃ isT or K; X₁₅₅ is S or T; X₁₆₃ is S or T; X₁₆₆ is Q, R, or H; X₁₇₂ is A orT; X₁₇₃ is A or V; X₁₇₄ is T or L; X₁₇₇ is A or E; X₁₇₈ is E or D; X₁₇₉is R, L, or K; X₁₈₀ is V or G; X₁₈₁ to X₁₈₄ inclusive is deleted, or isreplaced with the sequence S-(L or M)-(E or D)-H-R; X₁₈₅ is E; X₁₈₆ isY; X₁₈₇ is K or N; X₁₈₈ is K; X₁₉₀ is R or T; X₁₉₆ is G; X₂₀₁ is A or S;X₂₁₁ is V; X₂₁₂ is V or L; X₂₁₃ is D or E; X₂₁₅ is V or I; X₂₂₆ is S orR; X₂₄₄ is K or R; X₂₅₁ is Q or H; X₂₅₄ is V or I; X₂₅₅ is S or N; X₂₅₇is E or G; X₂₆₄ is T or A; X₂₇₄ is C or G; X₂₇₅ is V or I; X₂₈₀ is K orR; X₂₈₁ is S or N; X₂₈₅ is K or D; X₂₈₆ is K or R; X₂₈₇ is D or isdeleted; X₂₈₈ is R or is deleted; X₂₈₉ is I or L; X₂₉₁ is T or M; X₂₉₃is K or Q; X₂₉₇ is T or K; X₂₉₉ is I, T, or V; X₃₀₁ is R or H; X₃₀₃ is Nor D; X₃₀₅ is K; X₃₀₇ is R; X₃₀₉ is Q; X₃₁₁ is R; X₃₁₅ is Y or H; X₃₁₈is S or F; X₃₂₁ is E or D; X₃₂₆ is P or S; X₃₂₇ is C or L; and X₃₂₈ isS, G, or Q. As used herein and throughout the specification, each of thesingle letters in the amino acid sequences presented above represents aparticular amino acid residue, according to standard practice known tothose of ordinary skill in the art. In various embodiments, the modifiedp40 polypeptide encoded by the nucleic acid of the invention comprisesan amino acid sequence having at least about 90%, 92%, 95%, 96%, 97%,98%, or 99% amino acid sequence identity to the mature polypeptideregion (amino acid residue positions 23-328) of SEQ ID NO:39. In anotherembodiment, the nucleic acid of the invention encodes a modified p40polypeptide comprising an amino acid sequence identified herein as themature polypeptide region (amino acid residue positions 23-328) of SEQID NO:39.

The modified p40 polypeptide encoded by a nucleic acid of the inventionmay further comprise a leader peptide sequence having at least about90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to theamino acid sequence M-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identifiedherein as the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:39, where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅ isF or L; and X₂₁ is V or M.

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a polypeptide comprising aleader peptide sequence having at least about 90% amino acid sequenceidentity to the amino acid sequenceM-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identified herein as the leaderpeptide region (amino acid residue positions 1-22) of SEQ ID NO:39,where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅ is F or L; and X₂₁is V or M. In various embodiments, the leader peptide sequence encodedby the nucleic acid of the invention comprises an amino acid sequencehaving at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to leader peptide region (amino acid residues 1-22) ofSEQ ID NO:39.

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p35 polypeptidecomprising an amino acid modification located at an amino acid positionequivalent to (i.e., an equivalent position to) that in the amino acidsequence of a naturally-occurring (i.e., wild-type) p35 polypeptide (SEQID NO:36). The modification can include: (a) a substitution of thespecified amino acid for a different amino acid at one or moreequivalent position to that of SEQ ID NO:36 selected from Thr91, Met120,Ala121, Val212, Thr213, and Ala218; (b) a insertion of one or more aminoacids Phe-His-Leu between equivalent positions Leu19 and Ser20; (c) adeletion of the amino acid at equivalent position Pro36. The modifiedp35 polypeptide may optionally include two or more of modification (a),(b) or (c). The modified p35 polypeptide sequence encoded by the nucleicacid of the invention may be a modified sequence of anaturally-occurring (i.e., wild-type) p35 polypeptide sequence of amammal (e.g., human, a non-human primate, a ruminant, or a rodent),preferably a primate, more preferably human. The modified p35polypeptide sequence encoded by the nucleic acid of the invention may bea modified sequence of a polypeptide selected from the group consistingof p35 polypeptides encoded by nucleic acids having the GenBankaccession numbers M65271, M65291, U19842, U19835, U83185, Y07761,AF054605, U49085, L35765, Y11130, U14416, U57751, AF173557, AF003542,X97018, AF177031, M86672, and S82419. Preferred substitutions includeThr91(Ala or Ile); Met120Thr; Ala121Thr; Val212Met; Thr213Met; andAla218Ser.

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p35polypeptide, wherein the modified p35 polypeptide comprises an aminoacid sequence having at least about 90% amino acid sequence identity tothe amino acid sequence identified herein as the mature polypeptideregion (amino acid residue positions 23-219) of SEQ ID NO:40:R-X₂₄-LP-X₂₇-X₂₈-T-X₃₀-X₃₁-PG-X₃₄-X₃₅-X₃₆-CL-X₃₉-X₄₀-SQNLL-X₄₆-A-X₄₈-SN-X₅₁-LQ-X₅₄-A-X₅₆-Q-X₅₈-LEFY-X₆₃-CTSEE-X₆₉-DHEDIT-X₇₆-DKTSTVEACLPLEL-X₉₁-X₉₂-NESCL-X₉₈-SR-X₁₀₁-X₁₀₂-S-X₁₀₄-ITNGSCLASRKTSFM-X₁₂₀-X₁₂₁-LC-X₁₂₄-X₁₂₅-SIYEDLKMYQ-X₁₃₆-EFK-X₁₄₀-MNAKLLM-X₁₄₈-PKRQIFLDQNML-X₁₆₁-X₁₆₂-I-X₁₆₄-EL-X₁₆₇-QALN-X₁₇₂-NSET-X₁₇₇-PQK-X₁₈₁-SLEE-X₁₈₆-DFYKTKIKLCILLHAFRIRAVTI-X₂₁₀-R-X₂₁₂-X₂₁₃-SYLN-X₂₁₈-S,or a conservatively substituted variation thereof, where X₂₄ is N or S;X₂₇ is V or T; X₂₈ is A or T; X₃₀ is P or A; X₃₁ is D, S, or G; X₃₄ is Mor R; X₃₅ is F, S, or L; X₃₆ is P or is deleted; X₃₉ is H or D; X₄₀ is Hor Y; X₄₆ is R or K; X₄₈ is V or A; X₅₁ is M or T; X₅₄ is K or R; X₅₆ isK or R; X₅₈ is T or I; X₆₃ is P or S; X₆₉ is I or T; X₇₆ is K or Q; X₉₁is A or I; X₉₂ is K or T; X₉₈ is N or A; X₁₀₁ is E or G; X₁₀₂ is T or I;X₁₀₄ is F or L; X₁₂₀ is T; X₁₂₁ is T; X₁₂₄ is L or H; X₁₂₅ is S or G;X₁₃₆ is V or M; X₁₄₀ is T or A; X₁₄₈ is D or N; X₁₆₁ is A or T; X₁₆₂ isV or A; X₁₆₄ is D or A; X₁₆₇ is M or L; X₁₇₂ is F or V; X₁₇₇ is V or A;X₁₈₁ is S or P; X₁₈₆ is P or L; X₂₁₀ is D or N; X₂₁₂ is M; X₂₁₃ is M;and X₂₁₈ is S. In various embodiments, the nucleic acid of the inventionencodes a modified p35 polypeptide comprising an amino acid sequencehaving at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to the mature polypeptide region (amino acid residuepositions 23-219) of SEQ ID NO:40. In another embodiment, the nucleicacid of the invention encodes a modified p35 polypeptide comprising anamino acid sequence identified herein as the mature polypeptide region(amino acid residue positions 23-219) of SEQ ID NO:40.

The modified p35 polypeptide encoded by a nucleic acid of the inventionmay further comprise a leader peptide sequence having at least about90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to theamino acid sequence M-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₇-HLSL-X₂₂,identified herein as the leader peptide region (amino acid residuepositions 1-22) of SEQ ID NO:40, where X₂ is C or Y; X₄ is A, L or P; X₆is S or G; X₁₀ is V or I; X₁₁ is A or S; X₁₇ is D or H; and X₂₂ is A orG, and optionally includes an insertion of the amino acids P-H-L betweenpositions 18 and 19.

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a polypeptide comprising aleader peptide sequence having at least about 90% amino acid sequenceidentity to the amino acid sequenceM-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₆-HLSL-X₂₂, identified herein as theleader peptide region (amino acid residue positions 1-22) of SEQ IDNO:40, where X₂ is C or Y; X₄ is A, L, or P; X₆ is S or G; X₁₀ is V orI; X₁₁ is A or S; X₁₆ is D or H; X₂₂ is A or G, and optionally includesan insertion of the amino acids P-H-L between positions 18 and 19. Invarious embodiments, the leader peptide sequence encoded by the nucleicacid of the invention comprises an amino acid sequence having at leastabout 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identityto leader peptide region (amino acid residue positions 1-22) of SEQ IDNO:40.

The invention also provides nucleotide fragments of any of SEQ NOS:1-7.In one aspect of the invention, such a nucleotide fragment encodes apolypeptide comprising an amino acid sequence comprising at least 10contiguous amino acid residues of any one of SEQ ID NOS:8-14. Thepolypeptide sequence encoded by the nucleotide fragment typicallycomprises one or more amino acid substitution, at an equivalent positionto that of SEQ ID NO:15, selected from: Leu62Ser; Ser71Thr; Gln78His;His99(Arg or Gln); Thr127(Ser or Ile); Arg130Lys; Lys185Glu; Glu186Tyr;Tyr187(Lys or Asn); Glu188Lys; Ser190(Arg or Thr); As p196Gly;Met211Val; Val289(Ile or Leu); Ser305Lys; Ser307Arg; Arg309Gln; andGln311 Arg. The polypeptide may optionally comprises one or moresubstitution, at an equivalent position to that of SEQ ID NO:15,selected from: Cys2His; His3Pro; Ile8Val; Phe15Leu; Val21Met; Lys27Glu;Asp29Asn; Asp40Asn; Met45Thr; Thr49Ala; Glu67Gly, His91Arg; Glu95(Ala orThr), Val96Ala; Glu122Lys; Asn125Ala; Asn135Asp; Arg139His; Thr147Ala;Thr153Lys; Ser155Thr; Ser163Thr; Gln166(Arg or His), Ala172Thr;Ala173Val; Thr174Leu; Ala177Glu; Glu178Asp; Arg179Leu; Val180Gly;Ala201Ser; Val212Leu; Asp213Glu; Val215Ile; Ser226Arg; Lys244Arg;Gln251His; Val254Ile; Ser255Asn; Glu257Gly; Thr264(Ala or Ile);Thr272Met; Cys274Gly; Val275Ile; Lys280Arg; Ser281Asn; Lys285Asp;Lys286Arg; Phe290Ser; Thr291(Met or Val); Lys293Gln; Thr297Lys;Ile299(Thr or Val); Arg301His; Asn303Asp; Ser318Phe; Glu321Asp;Pro326Ser; Cys327Leu; and Ser328(Gly or Gln). In various embodiments,the nucleic acid of the invention encodes a polypeptide comprising atleast 10, at least 15, at least 20, at least 30, at least 50, at least75, at least 100, at least 150, at least 200, or at least 250 contiguousamino acid residues of any one of SEQ ID NOS:8-14. In other embodiments,the polypeptide encoded by the nucleic acid of the invention comprisesat least 270, 280, 285, 290, 295, 300, 305, 310, or 320 contiguous aminoacid residues of any one of SEQ ID NOS:8-14. In another embodiment, theencoded polypeptide comprises at least 290, 300, or 305 contiguous aminoacid residues of the mature polypeptide region of any one of SEQ IDNOS:8-14. In other embodiments, the invention provides a nucleic acidthat comprises a polynucleotide sequence selected from SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ IDNO:7.

The invention also provides nucleotide fragments of any of SEQNOS:16-25. In one aspect of the invention, such a nucleotide fragmentencodes a polypeptide comprising an amino acid sequence comprising atleast 10 contiguous amino acid residues of any one of SEQ ID NOS:26-35.The polypeptide typically comprises one or more amino acid substitution,at an equivalent position to that of SEQ ID NO:36, selected from:Thr91(Ala or Ile), Met120Thr, Ala121Thr, Val212Met, Thr213Met, andAla218Ser. The polypeptide optionally comprises one or moresubstitution, at an equivalent position to that of SEQ ID NO:36,selected from: Cys2Tyr; Ala4(Leu or Pro); Ser6Gly; Val10Ile; Ala11Ser;Asp17His; Ala22Gly; Asn24Ser; Val27Thr; Ala28Thr; Pro30Ala; Asp31(Ser orGly); Met34Arg; Phe35(Ser or Leu); Pro36(deleted); His39Asp; His40Tyr;Arg46Lys; Val48Ala; Met51Thr; Lys54Arg; Thr58Ile; Pro63Ser; Ile69Thr;Lys76Gln; Lys92Thr; Asn98Ala; Glu101Gly; Thr102Ile; Phe104Leu;Leu124His; Ser125Gly; Val136Met; Thr140Ala; Asp148Asn; Ala 161 Thr;Val162Ala; Asp164Ala; Met167Leu; Phe172Val; Val177Ala; Ser181Pro;Pro186Leu; Asp210Asn; and insertion of one or more of 220Leu; 221Glu;222Ser; and 223Ser. In various embodiments, the nucleic acid of theinvention encodes a polypeptide sequence comprising at least 10, atleast 15, at least 20, at least 30, at least 50, at least 75, at least100, at least 150, at least 170, or at least 180 contiguous amino acidresidues of any one of SEQ ID NOS:26-35. In another embodiment, theencoded polypeptide sequence comprises at least 185, 190, 195, 200, or205 contiguous amino acid residues of the mature polypeptide region ofany one of SEQ ID NOS:26-35. In other embodiments, the inventionprovides a nucleic acid that comprises a polynucleotide sequenceselected from SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,and SEQ ID NO:25.

An isolated or recombinant polypeptide having a sequence encoded by anynucleic acid of the invention, such as nucleic acids embodied in SEQ IDNOS:1-7 and SEQ ID NOS:16-25, is also a feature of the invention.

An isolated or recombinant polypeptide having any of the precedingsequences, such as those embodied in SEQ ID NOS:8-14, SEQ ID NO:39, SEQID NOS:26-35, and SEQ ID NO:40, is also a feature of the invention.

The invention also provides polypeptide fragments of any of SEQNOS:8-14, SEQ ID NO:39, and SEQ ID NOS:26-35, SEQ ID NO:40. In oneaspect of the invention, such a polypeptide fragment exhibits T-cellproliferative activity or interferon-gamma induction activity in aT-cell based assay (such as, e.g., a human T-cell based assay). TheT-cell proliferation assay and the interferon-gamma induction assay aredescribed in greater detail below. In yet another aspect, the inventionprovides a polynucleotide sequence comprising a nucleotide fragment ofany nucleic acid of the invention described above and below, whereinsaid nucleotide fragment encodes a polypeptide fragment that exhibitsT-cell proliferative activity or interferon-gamma induction activity ina T-cell based assay (such as, e.g., a human T-cell based assay), as isdescribed in greater detail below.

In other embodiments, the modified p40 polypeptide of the invention canform a dimer (i.e., the polypeptide “dimerizes”) with a p35 polypeptideto form a modified p40/p35 heterodimer, or can dimerize with a p40polypeptide to form a modified p40/p40 homodimer. In variousembodiments, the modified p40 polypeptide of the invention(hetero-)dimerizes with a polypeptide comprising a naturally-occurringor “wild type” p35 polypeptide sequence (e.g., a human p35 polypeptide,such as SEQ ID NO:36; a primate p35 polypeptide; or a mammalian p35polypeptide), or a fragment thereof. In other embodiments, the modifiedp40 polypeptide of the invention (hetero-)dimerizes with a modified p35polypeptide of the invention, such as, for example, one comprising asequence selected from SEQ ID NOS:26-35, SEQ ID NO:40, or a fragmentthereof. In various other embodiments, the modified p40 polypeptide ofthe invention (homo-)dimerizes with a polypeptide comprising anaturally-occurring or wild-type p40 polypeptide sequence (e.g., a humanp40 polypeptide, such as SEQ ID NO:15, a primate p40 polypeptide, or amammalian p40 polypeptide), or a fragment thereof. In other embodiments,the modified p40 polypeptide of the invention (homo-)dimerizes withitself, or with another modified p40 polypeptide of the invention, suchas, for example, one comprising a sequence selected from SEQ ID NOS:8-14or SEQ ID NO:39, or a fragment thereof.

In other embodiments, the modified p35 polypeptide of the inventionforms a dimer (i.e., the polypeptide “dimerizes”) with a p40 polypeptideto form a modified p40/p35 heterodimer, or with a p35 polypeptide toform a modified p35/p35 homodimer. In various embodiments, the modifiedp35 polypeptide of the invention (hetero-)dimerizes with a polypeptidecomprising a naturally-occurring or wild-type p40 polypeptide sequence(e.g., a human p40 polypeptide, such as SEQ ID NO:15, a primate p40polypeptide, or a mammalian p40 polypeptide), or a fragment thereof. Inother embodiments, the modified p35 polypeptide of the invention(hetero-)dimerizes with a modified p40 polypeptide of the invention,such as, for example, one comprising a sequence selected from SEQ IDNOS:8-14 and SEQ ID NO:39, or a fragment thereof. In various otherembodiments, the modified p35 polypeptide of the invention(homo-)dimerizes with a polypeptide comprising a naturally-occurring orwild-type p35 polypeptide sequence (e.g., a human p35 polypeptide, suchas SEQ ID NO:36, a primate p35 polypeptide, or a mammalian p35polypeptide), or a fragment thereof. In other embodiments, the modifiedp35 polypeptide of the invention (homo-) dimerizes with itself, or withanother modified p35 polypeptide of the invention, such as, for example,one comprising a sequence selected from SEQ ID NOS:26-35 and SEQ IDNO:40, or a fragment thereof.

The invention also includes a cell comprising any nucleic acid of theinvention described herein, or which expresses any polypeptide of theinvention noted herein. In one embodiment, the cell expresses apolypeptide encoded by the nucleic acid of the invention as describedherein.

The invention also includes a vector comprising any nucleic acid of theinvention described above and below. The vector can comprise a plasmid,a cosmid, a phage, or a virus; the vector can be, e.g., an expressionvector, a cloning vector, a packaging vector, an integration vector, orthe like. The invention also includes a cell transduced by a vector ofthe invention. The invention also includes compositions comprising anynucleic acid of the invention described above and below, and anexcipient, preferably a pharmaceutically acceptable excipient. Cells andtransgenic animals which include any polypeptide or nucleic acid of theinvention described above and below, e.g., produced by transduction ofvector, are a feature of the invention.

The invention also includes compositions produced by digesting one ormore nucleic acid described above with, e.g., a restrictionendonuclease, an RNAse, or a DNAse; compositions produced by fragmentingone or more nucleic acid described above by mechanically shearing, byUV, or by chemical methods; and compositions produced by incubating oneor more nucleic acid described above in the presence of ribonucleotideor deoxyribonucelotide triphosphates and a nucleic acid polymerase,e.g., a thermostable polymerase.

The invention also includes compositions comprising two or more nucleicacids described above. The composition may comprise a library of nucleicacids, where the library contains, e.g., at least 2, 3, 5, 10, 20 or 50nucleic acids.

In another aspect, the invention includes an isolated or recombinantpolypeptide encoded by any nucleic acid of the invention describedherein. In one embodiment, the polypeptide may comprise a maturepolypeptide region of a sequence selected from SEQ ID NO:8 to SEQ IDNO:14 and SEQ ID NO:39, or SEQ ID NO:26 to SEQ ID NO:35 and SEQ IDNO:40. Longer polypeptides; e.g., which comprise leader peptidesequences, purification tags, or the like, are also contemplated. Suchpolypeptides may display T-cell proliferative activity and/orinterferon-gamma induction activity in a T-cell based assay (such as,e.g., a human T-cell based assay).

The invention also includes a polypeptide which specifically bindspolyclonal antisera raised against at least one antigen, the at leastone antigen comprising a polypeptide sequence selected from an aminoacid sequence set forth in SEQ ID NO:8 to SEQ ID NO:14 or SEQ ID NO:26to SEQ ID NO:35 or a fragment thereof. In particular, the inventionprovides polypeptides which bind a polyclonal antisera raised against atleast one antigen, wherein said at least one antigen comprises at leastone amino acid sequence set forth in SEQ ID NO: 8 to SEQ ID NO:14, or afragment of any of these amino sequences, wherein the polyclonalantisera is subtracted with one or more known p40 polypeptides orproteins, including, e.g., a polypeptide or protein encoded by a nucleicacid having or corresponding to one or more of the following GenBank™accession numbers: M65272 and M65290 (human), U19841 (Macaca mulatta,rhesus monkey), U19834 (Cercocebus torquatus, sooty mangabey), Y11129(Equus caballus, horse), U83184, Y07762 and AF054607 (Felis catus, cat),U49100 and AF091134 (Canis familiaris, dog), U57752 and U10160 (Cervuselaphus, red deer), AF007576 (Capra hircus, goat), AF004024 (Ovis aries,sheep), U11815 (Bos taurus, cow), U08317 (Sus scrofa, pig), X97019 andAF082494 (Marmota monax, woodchuck), AF133197 and U16674 (Rattusnorvegicus, rat), M86671 and S82426 (Mus musculus, mouse), AF097507(Cavia porcellus, guinea pig), and AF046211 (Mesocricetus auratus,golden hamster), and other similar or homologous p40 nucleic acidsequences presented in, e.g., GenBank

The invention also provides polypeptides which bind a polyclonalantisera raised against at least one antigen, wherein said at least oneantigen comprises at least one amino acid sequence set forth in SEQ IDNO:26 to SEQ ID NO:35, or a fragment of any of these amino sequences,wherein the polyclonal antisera is subtracted with one or more known p35polypeptides or proteins, including, e.g., a polypeptide or proteinencoded by a nucleic acid having or corresponding to one or more of thefollowing GenBank accession numbers: M65271, M65291 (human); U19842(Macaca mulatta, rhesus monkey), U19835 (Cercocebus torquatus, sootymangabey), U83185, Y07761, AF054605 (Felis catus, cat), U49085 (Canisfamiliaris, dog), L35765 (Sus scrofa, pig), Y11130 (Equus caballus,horse), U14416 (Bos taurus, cow), U57751 (Cervus elaphus, red deer),AF173557 (Ovis aries, sheep), AF003542 (Capra hircus, goat), X97018(Marmota monax, woodchuck), AF177031 (Rattus norvegicus, rat), andM86672, S82419 (Mus musculus, mouse), and other similar or homologousp35 nucleic acid sequences presented in, e.g., GenBank.

As described above, a polypeptide of the invention may form a dimer(e.g., a heterodimer or a homodimer) with either a p35 polypeptide or ap40 polypeptide; A composition comprising a polypeptide of the invention(alone or in combination with an additional p35 polypeptide or p40polypeptide) may exhibit at least one of the following activities: (a)T-cell proliferative activity, (b) IFN-γ induction activity, (c)enhancement of NK cell-mediated toxicity. The p35 polypeptide maycomprise a naturally-occurring or wild-type sequence, such as SEQ IDNO:36 or a fragment thereof, or may comprise a modified sequence, suchas, for example, one of SEQ ID NO:26 to 35, SEQ ID NO:40, or a fragmentthereof. Likewise, the p40 polypeptide may comprise anaturally-occurring or wild-type sequence, such as SEQ ID NO:15 or afragment thereof, or may comprise a modified sequence, such as, forexample, one of SEQ ID NO:8 to 14, SEQ ID NO:39, or a fragment thereof.

In other embodiments, any polypeptide described above may furtherinclude a secretion/localization sequence, e.g., a leader (or signal)peptide sequence, an organelle targeting sequence, a membranelocalization sequence, and the like. Any polypeptide described above mayfurther include a sequence that facilitates purification, e.g., anepitope tag (such as, a FLAG epitope or an E-Tag epitope), apolyhistidine tag, a GST fusion, and the like. The polypeptideoptionally includes a methionine at the N-terminus. Any polypeptidedescribed above optionally includes one or more modified amino acid,such as a glycosylated amino acid, a PEG-ylated amino acid, afarnesylated amino acid, an acetylated amino acid, a biotinylated aminoacid, a carboxylated amino acid, a phosphorylated amino acid, anacylated amino acid, or the like.

The invention also includes compositions comprising any nucleic acid orpolypeptide described above in an excipient, preferably apharmaceutically acceptable excipient.

The invention also includes an antibody or antisera produced byadministering one or more of the polypeptides of the invention describedherein to a mammal, wherein the antibody or antisera does notspecifically bind to a known p40 polypeptide or protein, including,e.g., any polypeptide or protein encoded by a nucleic acid having orcorresponding to one or more of the following GenBank accession numbers:M65272 and M65290 (human), U19841 (Macaca mulatta, rhesus monkey),U19834 (Cercocebus torquatus, sooty mangabey), Y11129 (Equus caballus,horse), U83184, Y07762 and AF054607 (Felis catus, cat), U49100 andAF091134 (Canis familiaris, dog), U57752 and U10160 (Cervus elaphus, reddeer), AF007576 (Capra hircus, goat), AF004024 (Ovis aries, sheep),U11815 (Bos taurus, cow), U08317 (Sus scrofa, pig), X97019 and AF082494(Marmota monax, woodchuck), AF133197 and U16674 (Rattus norvegicus,rat), M86671 and S82426 (Mus musculus, mouse), AF097507 (Caviaporcellus, guinea pig), and AF046211 (Mesocricetus auratus, goldenhamster), and other similar or homologous p40 sequences presented in,e.g., GenBank. The invention also includes antibodies or antiseraproduced by administering one or more of the polypeptides of theinvention described herein to a mammal, wherein the antibody or antiseradoes not bind to a known p35 polypeptide or protein, including, e.g.,any polypeptide or protein encoded by a nucleic acid having orcorresponding to one or more of the following GenBank accession numbers:M65271, M65291 (Homo sapiens); U19842 (Macaca mulatta, rhesus monkey),U19835 (Cercocebus torquatus, sooty mangabey), U83185, Y07761, AF054605(Felis catus, cat), U49085 (Canis familiaris, dog), L35765 (Sus scrofa,pig), Y11130 (Equus caballus, horse), U14416 (Bos taurus, cow), U57751(Cervus elaphus, red deer), AF173557 (Ovis aries, sheep), AF003542(Capra hircus, goat), X97018 (Marmota monax, woodchuck), AF177031(Rattus norvegicus, rat), and M86672, S82419 (Mus musculus, mouse), andother similar or homologous p35 sequences presented in, e.g., GenBank.

The invention also includes antibodies which specifically bind apolypeptide comprising a sequence selected from SEQ ID NO:8 to SEQ IDNO:14 or SEQ ID NO:26 to SEQ ID NO:35. The antibodies are, e.g.,polyclonal, monoclonal, chimeric, humanized, single chain, Fabfragments, fragments produced by an Fab expression library, or the like.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any nucleic acid of the invention described above, operativelylinked to a regulatory sequence effective to produce the encodedpolypeptide, culturing the cells in a culture medium to produce thepolypeptide, and optionally isolating the polypeptide from the cells orfrom the culture medium. The nucleic acid may be part of a vector, suchas a recombinant expression vector.

The invention also includes compositions comprising a nucleic acid ofthe invention, and optionally, further comprising a second nucleic acidencoding a p35 polypeptide or a p40 polypeptide. The second nucleic acidmay encode a p35 polypeptide having a naturally-occurring p35 sequence,such as a nucleic acid encoding SEQ ID NO:36, or may encode a modifiedp35 polypeptide sequence, such as, for example, one of nucleic acids SEQID NOS:16-25 encoding SEQ ID NOS:26-35, respectively. The second nucleicacid may encode a p40 polypeptide having a naturally-occurring sequence,such as a nucleic acid encoding SEQ ID NO:15, or may encode a modifiedp40 polypeptide sequence, such as, for example, one of nucleic acids SEQID NOS:1-7 encoding SEQ ID NOS:8-14, respectively.

The invention also includes compositions comprising a polypeptide of theinvention, and optionally, further comprising a second p35 polypeptideor a second p40 polypeptide. The second p35 polypeptide or the secondp40 polypeptide may have a naturally-occurring or wild-type sequence, ormay have a modified sequence, such as a modified p35 polypeptide or amodified p40 polypeptide of the invention.

The invention also includes a method of inducing proliferation ofT-cells comprising contacting the T-cells with a composition comprisinga polypeptide of the invention, thereby inducing proliferation of theT-cells. In one embodiment, the T-cells are in culture. In anotherembodiment, the T-cells are human T-cells.

The invention also includes a method of inducing production of IFN-γ inT-cells, the method comprising contacting the T-cells with a compositioncomprising a polypeptide of the invention, thereby inducing productionof IFN-γ in the T-cells. In one embodiment, the T-cells are in culture.In another embodiment, the T-cells are human T-cells.

In general, nucleic acids and proteins derived by mutation of thesequences herein are a feature of the invention. Similarly, thoseproduced by diversity generation methods or recursive sequencerecombination (“RSR”) methods (e.g., DNA shuffling) are a feature of theinvention. Mutation and recombination methods using the nucleic acidsdescribed herein are a feature of the invention. For example, one methodof the invention includes recursively recombining one or more nucleicacid sequences of the invention as described above and below with one ormore additional nucleic acids (including, but not limited to, thosenoted herein), each sequence of the one or more additional nucleic acidsencoding a modified p40 polypeptide or modified p35 polypeptide or anamino acid subsequence thereof. The recombining steps are optionallyperformed in vivo, ex vivo, in silico or in vitro. Said diversitygeneration or recursive sequence recombination produces at least onelibrary of recombinant modified p40 or modified p35 nucleic acids. Alsoincluded in the invention are a recombinant modified p40 nucleic acidproduced by this method, a recombinant modified p35 nucleic acidproduced by this method, a cell containing the recombinant modified p40nucleic acid or recombinant modified p35 nucleic acid, a nucleic acidlibrary produced by recursive sequence recombination or other diversitygeneration methods, a composition comprising two or more of recombinantmodified p40 or modified p35 nucleic acids, and a population of cellscomprising such recombinant modified p40 or modified p35 nucleic acidsor containing the library. In one embodiment, the library comprise atleast ten such recombinant nucleic acids.

The invention also provides a method of producing a modified orrecombinant modified p40 or modified p35 nucleic acid that comprisesmutating a nucleic acid of the invention as described herein.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show an alignment of a naturally-occurring or wild-typehuman p40 polypeptide sequence (SEQ ID NO:15) and exemplary modified p40polypeptide sequences according to the invention (SEQ ID NOS:8-14).Equivalent amino acid residue positions which differ from those of SEQID NO:15 are shaded. The arrow and the dashed horizontal line, locatedbetween the amino acid residue positions equivalent to amino acidresidues 22 and 23 of SEQ ID NO:15, indicate the predicted boundarybetween the leader peptide region and the mature polypeptide region. Thealignment was prepared using the CLUSTALW multiple sequence alignmentalgorithm, a part of the Vector NTI version 6 sequence analysis softwarepackage (Informax, Bethesda, Md.). The CLUSTALW program initiallyperforms multiple pairwise comparisons between groups of sequences andthen assembles the pairwise alignments into a multiple alignment basedon homology. For the initial pairwise alignments, Gap Open and GapExtension penalties were 10 and 0.1, respectively. For the multiplealignments, Gap Open penalty was 10, and the Gap Extension penalty was0.05. The protein weight matrix employed was the BLOSUM62 matrix.

FIG. 2 shows T-cell proliferative activity of two-fold serial dilutionsof culture media of mammalian cell cultures expressing a modified p40nucleic acid of the invention (SEQ ID NO:7 encoding R16-51) co-expressedwith a nucleic acid comprising a wild-type human p35 (“wt-p35”) codingsequence (SEQ ID NO:38), as compared to equivalent serial dilutions of acontrol culture co-expressing nucleic acids comprising wt-p35 andwild-type human p40 (“wt-p40”) coding sequences (SEQ ID NO:38 and SEQ IDNO:37, respectively). The X-axis (“Fold-Dilution”) indicates the n-foldserial dilution of culture supernatant, and the Y-axis (“CPM”) indicatesthe amount of ³H-thymidine incorporated into T-cells, expressed ascounts per minute. For this and the following figures, the data barsrepresents the average, and the error bars represent the standarddeviation, of at least three data points per assay.

FIG. 3 shows T-cell proliferative activities of two-fold serialdilutions of culture media of mammalian cell cultures expressing thefollowing modified p40 nucleic acids of the invention: SEQ ID NO:6encoding A16-94; SEQ ID NO:2 encoding B8-96; and SEQ ID NO:1 encodingC2-22, all co-expressed with a nucleic acid comprising a wt-p35 codingsequence (SEQ ID NO:37), as compared to equivalent serial dilutions of acontrol culture co-expressing nucleic acids comprising wt-p35 and wt-p40coding sequences (SEQ ID NO:38 and SEQ ID NO:37, respectively). X- andY-axes are as described for FIG. 2.

FIG. 4 shows T-cell proliferative activity of two-fold serial dilutionsof culture media of mammalian cell cultures expressing a modified p40nucleic acid of the invention (SEQ ID NO:5 encoding A3-48) co-expressedwith a nucleic acid comprising a wt-p35 coding sequence, as compared toequivalent dilutions of a control culture co-expressing nucleic acidscomprising wt-p35 and wt-p40 coding sequences (SEQ ID NO:38 and SEQ IDNO:37, respectively). X- and Y-axes are as described for FIG. 2.

FIG. 5 shows T-cell proliferative activities of two-fold serialdilutions of culture media of mammalian cell cultures expressing amodified p40 nucleic acid of the invention (SEQ ID NO:3 encoding B2-52)co-expressed with a nucleic acid comprising a wt-p35 coding sequence(SEQ ID NO:38), as compared to equivalent dilutions of a control cultureco-expressing nucleic acids comprising wt-p35 and wt-p40 codingsequences (SEQ ID NO:38 and SEQ ID NO:37, respectively). X- and Y-axesare as described for FIG. 2.

FIG. 6 shows T-cell proliferative activity of two-fold serial dilutionsof culture media of mammalian cell cultures expressing a modified p40nucleic acid of the invention (SEQ ID NO:4 encoding B1-81) co-expressedwith a nucleic acid comprising a wt-p35 coding sequence (SEQ ID NO:38),as compared to equivalent dilutions of a control culture co-expressingnucleic acids comprising wt-p35 and wt-p40 coding sequences (SEQ IDNO:38 and SEQ ID NO:37, respectively). X- and Y-axes are as describedfor FIG. 2.

FIG. 7 shows an immunoblot, probed using an anti-p35 monoclonal antibody(mAB), of equal volumes of cell culture supernatant from a control cellculture expressing wt-p40/wt-p35 nucleic acids, and from test cellcultures expressing A16-94/wt-p35, A3-48/wt-p35, B1-81/wt-p35,B2-52/wt-p35, B8-96/wt-p35 and C2-22/wt-p35 nucleic acids. Bandscorresponding to ˜70 kilodalton (kDa) heterodimeric proteins areindicated by the arrow labeled “p70” and bands corresponding to ˜35 kDap35 polypeptides are indicated by the arrow labeled “p35”. “MW”indicates molecular weight markers.

FIG. 8 shows an immunoblot, probed using an anti-p35 mAB, of dilutionsof purified C2-22/wt-p35 and wt-p40/wt-p35 heterodimers. The X-axisshows the volume of purified protein solution applied to the gel, andthe Y-axis shows the approximate molecular weight (in kDa).

FIG. 9 shows the T-cell proliferative activity of varying concentrationsof purified C2-22/wt-p35 and wt-p40/wt-p35 heterodimers. The X-axisindicates the concentration of heterodimer assayed, in nanograms permilliliter (ng/ml). The Y-axis (“CPM”) indicates the amount of³H-thymidine incorporated into T-cells, expressed as counts per minute.

FIG. 10 shows the IFN-γ induction activity of varying concentrations ofpurified C2-22/wt-p35 and wt-p40/wt-p35 heterodimers and a commerciallyavailable cytokine protein. The X-axis indicates the concentration ofheterodimer assayed, in nanograms per milliliter (ng/ml). The Y-axisindicates the amount of IFN-γ produced in T-cells, expressed aspicograms per milliliter (pg/ml).

FIGS. 11A-11B show an alignment of a naturally-occurring or wild-typehuman p35 polypeptide sequence (SEQ ID NO:36) and exemplary modified p35polypeptide sequences according to the invention (SEQ ID NOS:26-35).Equivalent amino acid positions which differ from those of SEQ ID NO:36are shaded. The arrow and the dashed horizontal line, located betweenthe amino acid residue positions equivalent to amino acid residues 22and 23 of SEQ ID NO:36, indicate the predicted boundary between theleader peptide region and the mature polypeptide region. The alignmentwas prepared using the CLUSTALW algorithm and Vector NTI software asdescribed in FIG. 1.

FIG. 12 shows the T-cell proliferative activity of varyingconcentrations of purified “fully modified” heterodimerC2-22/R2-571-EHtag and control heterodimer wt-p40/wt-p35-EHtag. X- andY-axes are as described in FIG. 9.

FIG. 13 shows the IFN-γ induction activity of varying concentrations ofpurified fully modified heterodimer C2-22/R2-571-EHtag and controlheterodimer wt-p40/wt-p35-EHtag. X- and Y-axes are as described in FIG.10.

FIG. 14 shows a Western blot, probed using an anti-E tag mAB(Pharmacia-Amersham), of purified heterodimers produced by co-expressinga modified p40 nucleic acid of the invention comprising the C2-22polynucleotide sequence SEQ ID NO:1 plus a modified p35 nucleic acid ofthe invention comprising the R2-571 polynucleotide sequence SEQ IDNO:21, and purified heterodimers produced by co-expressing wt-p40 andwt-p35 nucleic acids comprising the polynucleotide sequences SEQ IDNO:37 and SEQ ID NO:38, respectively. The modified p35 nucleic acidsequence and the wt-p35 nucleic acid sequence each further comprised aC-terminal EH-tag coding sequence. The modified heterodimer preparation,indicated on the figure as “C2-22/R2-571”, was loaded onto the gel inserial dilutions as indicated over each lane. The wt-p40/wt-p35heterodimer preparation, indicated on the figure as “wt”, was loaded ata single dilution. Bands corresponding to ˜70 kDa heterodimeric proteinsare indicated by the arrow labeled “p70” and bands corresponding to ˜35kDa p35 polypeptides are indicated by the arrow labeled “p35”. Highermolecular weight proteins which immunoreact with the anti-E tag antibodyare indicated by the bracket labeled “multimeric forms”. “MW” indicatesmolecular weight markers.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the present invention belongs.

A “polynucleotide sequence” is a nucleic acid (which is a polymer ofnucleotides (A,C,T,U,G, etc. or naturally occurring or artificialnucleotide analogues) or a character string representing a nucleic acid,depending on context. Either the given nucleic acid or the complementarynucleic acid can be determined from any specified polynucleotidesequence.

Similarly, an “amino acid sequence” is a polymer of amino acids (aprotein, polypeptide, etc.) or a character string representing an aminoacid polymer, depending on context. Either the given nucleic acid or thecomplementary nucleic acid can be determined from any specifiedpolynucleotide sequence.

A nucleic acid, protein, peptide, polypeptide, or other component is“isolated” when it is partially or completely separated from componentswith which it is normally associated (other peptides, polypeptides,proteins (including complexes, e.g., polymerases and ribosomes which mayaccompany a native sequence), nucleic acids, cells, synthetic reagents,cellular contaminants, cellular components, etc.), e.g., such as fromother components with which it is normally associated in the cell fromwhich it was originally derived. A nucleic acid, polypeptide, or othercomponent is isolated when it is partially or completely recovered orseparated from other components of its natural environment such that itis the predominant species present in a composition, mixture, orcollection of components (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In preferredembodiments, the preparation consists of more than 70%, typically morethan 80%, or preferably more than 90% of the isolated species.

In one aspect, a “substantially pure” or “isolated” nucleic acid (e.g.,RNA or DNA), polypeptide, protein, or composition also means where theobject species (e.g., nucleic acid or polypeptide) comprises at leastabout 50, 60, or 70 percent by weight (on a molar basis) of allmacromolecular species present. A substantially pure or isolatedcomposition can also comprise at least about 80, 90, or 95 percent byweight of all macromolecular species present in the composition. Anisolated object species can also be purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of derivatives of a single macromolecular species.

The term “isolated nucleic acid” may refer to a nucleic acid (e.g., DNAor RNA) that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (i.e., one at the 5′and one at the 3′ end) in the naturally occurring genome of the organismfrom which the nucleic acid of the invention is derived. Thus, this termincludes, e.g., a cDNA or a genomic DNA fragment produced by polymerasechain reaction (PCR) or restriction endonuclease treatment, whether suchcDNA or genomic DNA fragment is incorporated into a vector, integratedinto the genome of the same or a different species than the organism,including, e.g., a virus, from which it was originally derived, linkedto an additional coding sequence to form a hybrid gene encoding achimeric polypeptide, or independent of any other DNA sequences. The DNAmay be double-stranded or single-stranded, sense or antisense.

A nucleic acid or polypeptide is “recombinant” when it is artificial orengineered, or derived from an artificial or engineered protein ornucleic acid. The term “recombinant” when used with reference e.g., to acell, nucleotide, vector, or polypeptide typically indicates that thecell, nucleotide, or vector has been modified by the introduction of aheterologous (or foreign) nucleic acid or the alteration of a nativenucleic acid, or that the polypeptide has been modified by theintroduction of a heterologous amino acid, or that the cell is derivedfrom a cell so modified. Recombinant cells express nucleic acidsequences (e.g., genes) that are not found in the native(non-recombinant) form of the cell or express native nucleic acidsequences (e.g., genes) that would be abnormally expressedunder-expressed, or not expressed at all. The term “recombinant nucleicacid” (e.g., DNA or RNA) molecule means, for example, a nucleotidesequence that is not naturally occurring or is made by the combatant(for example, artificial combination) of at least two segments ofsequence that are not typically included together, not typicallyassociated with one another, or are otherwise typically separated fromone another. A recombinant nucleic acid can comprise a nucleic acidmolecule formed by the joining together or combination of nucleic acidsegments from different sources and/or artificially synthesized. Theterm “recombinantly produced” refers to an artificial combinationusually accomplished by either chemical synthesis means, recursivesequence recombination of nucleic acid segments or other diversitygeneration methods (such as, e.g., shuffling) of nucleotides, ormanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques known to those of ordinary skill in the art.“Recombinantly expressed” typically refers to techniques for theproduction of a recombinant nucleic acid in vitro and transfer of therecombinant nucleic acid into cells in vivo, in vitro, or ex vivo whereit may be expressed or propagated. A “recombinant polypeptide” or“recombinant protein” usually refers to polypeptide or protein,respectively, that results from a cloned or recombinant gene or nucleicacid.

A “subsequence” or “fragment” is any portion of an entire sequence, upto and including the complete sequence.

Numbering of a given amino acid or nucleotide polymer “corresponds to(the) numbering” of a selected amino acid polymer or nucleic acid whenthe position of any given polymer component (amino acid residue,incorporated nucleotide, etc.) is designated by reference to the sameresidue position in the selected amino acid or nucleotide, rather thanby the actual position of the component in the given polymer.

A vector is a composition for facilitating cell transduction by aselected nucleic acid, or expression of the nucleic acid in the cell.Vectors include, e.g., plasmids, cosmids, viruses, YACs, bacteria,poly-lysine, etc. An “expression vector” is a nucleic acid construct,generated recombinantly or synthetically, with a series of specificnucleic acid elements that permit transcription of a particular nucleicacid in a host cell. The expression vector can be part of a plasmid,virus, or nucleic acid fragment. The expression vector typicallyincludes a nucleic acid to be transcribed operably linked to a promoter.

“Substantially an entire length of a polynucleotide or amino acidsequence” refers to at least about 50%, at least about 60%, generally atleast about 70%, generally at least about 80%, or typically at leastabout 85%, 90%, 92%, 95,%, 96%, 97%, 98%, or 99% or more of a length ofan amino acid sequence or nucleic acid sequence.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism, includingviruses, that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory is naturallyoccurring.

As used herein, an “antibody” refers to a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chains,respectively. Antibodies exist as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include single chainantibodies, including single chain Fv (sFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

An “antigen-binding fragment” of an antibody is a peptide or polypeptidefragment of the antibody which binds an antigen. An antigen-binding siteis formed by those amino acids of the antibody which contribute to, areinvolved in, or affect the binding of the antigen. See Scott, T. A. andMercer, E. I., CONCISE ENCYCLOPEDIA: BIOCHEMISTRY AND MOLECULAR BIOLOGY(de Gruyter, 3d ed. 1997) [hereinafter “Scott, CONCISE ENCYCLOPEDIA”]and Watson, J. D. et al., RECOMBINANT DNA (2d ed. 1992) [hereinafter“Watson, RECOMBINANT DNA”], each of which is incorporated herein byreference in its entirety for all purposes.

An “immunogen” refers to a substance that is capable of provoking animmune response. Examples of immunogens include, e.g., antigens,autoantigens that play a role in induction of autoimmune diseases, andtumor-associated antigens expressed on cancer cells.

An “antigen” is a substance that is capable of eliciting the formationof antibodies in a host or generating a specific population oflymphocytes reactive with that substance. Antigens are typicallymacromolecules (e.g., proteins and polysaccharides) that are foreign tothe host.

The term “immunoassay” includes an assay that uses an antibody orimmunogen to bind or specifically bind an antigen. The immunoassay istypically characterized by the use of specific binding properties of aparticular antibody to isolate, target, and/or quantify the antigen.

The term “homology” generally refers to the degree of similarity betweentwo or more structures. The term “homologous sequences” refers toregions in macromolecules that have a similar order of monomers. Whenused in relation to nucleic acid sequences, the term “homology” refersto the degree of similarity between two or more nucleic acid sequences(e.g., genes) or fragments thereof. Typically, the degree of similaritybetween two or more nucleic acid sequences refers to the degree ofsimilarity of the composition, order, or arrangement of two or morenucleotide bases (or other genotypic feature) of the two or more nucleicacid sequences. The term “homologous nucleic acids” generally refers tonucleic acids comprising nucleotide sequences having a degree ofsimilarity in nucleotide base composition, arrangement, or order. Thetwo or more nucleic acids may be of the same or different species orgroup. The term “percent homology” when used in relation to nucleic acidsequences, refers generally to a percent degree of similarity betweenthe nucleotide sequences of two or more nucleic acids.

When used in relation to polypeptide (or protein) sequences, the term“homology” refers to the degree of similarity between two or morepolypeptide (or protein) sequences (e.g., genes) or fragments thereof.Typically, the degree of similarity between two or more polypeptide (orprotein) sequences refers to the degree of similarity of thecomposition, order, or arrangement of two or more amino acid of the twoor more polypeptides (or proteins). The two or more polypeptides (orproteins) may be of the same or different species or group. The term“percent homology” when used in relation to polypeptide (or protein)sequences, refers generally to a percent degree of similarity betweenthe amino acid sequences of two or more polypeptide (or protein)sequences. The term “homologous polypeptides” or “homologous proteins”generally refers to polypeptides or proteins, respectively, that haveamino acid sequences and functions that are similar. Such homologouspolypeptides or proteins may be related by having amino acid sequencesand functions that are similar, but are derived or evolved fromdifferent or the same species using the techniques described herein.

The term “subject” as used herein includes, but is not limited to, anorganism; a mammal, including, e.g., a human, non-human primate (e.g.,monkey), mouse, dog, cat, pig, cow, goat, rabbit, rat, guinea pig,hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal,including, e.g., a non-mammalian vertebrate, such as a bird (e.g., achicken or duck) or a fish; and a non-mammalian invertebrate.

The term “pharmaceutical composition” means a composition suitable forpharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount of anactive agent and a pharmaceutically acceptable carrier.

The term “effective amount” means a dosage or amount sufficient toproduce a desired result. The desired result may comprise an objectiveor subjective improvement in the recipient of the dosage or amount.

A “prophylactic treatment” is a treatment administered to a subject whodoes not display signs or symptoms of a disease, pathology, or medicaldisorder, or displays only early signs or symptoms of a disease,pathology, or disorder, such that treatment is administered for thepurpose of diminishing, preventing, or decreasing the risk of developingthe disease, pathology, or medical disorder. A prophylactic treatmentfunctions as a preventative treatment against a disease or disorder. A“prophylactic activity” is an activity of an agent, such as a nucleicacid, vector, gene, polypeptide, protein, substance, composition thereofthat, when administered to a subject who does not display signs orsymptoms of pathology, disease or disorder, or who displays only earlysigns or symptoms of pathology, disease, or disorder, diminishes,prevents, or decreases the risk of the subject developing a pathology,disease, or disorder. A “prophylactically useful” agent or compound(e.g., nucleic acid or polypeptide) refers to an agent or compound thatis useful in diminishing, preventing, treating, or decreasingdevelopment of pathology, disease or disorder.

A “therapeutic treatment” is a treatment administered to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms of pathology, disease, ordisorder. A “therapeutic activity” is an activity of an agent, such as anucleic acid, vector, gene, polypeptide, protein, substance, orcomposition thereof, that eliminates or diminishes signs or symptoms ofpathology, disease or disorder, diminishes when administered to asubject suffering from such signs or symptoms. A “therapeuticallyuseful” agent or compound (e.g., nucleic acid or polypeptide) indicatesthat an agent or compound is useful in diminishing, treating, oreliminating such signs or symptoms of a pathology, disease or disorder.

The term “gene” broadly refers to any segment of DNA associated with abiological function. Genes include coding sequences and/or regulatorysequences required for their expression. Genes also includenon-expressed DNA nucleic acid segments that, e.g., form recognitionsequences for other proteins.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, molecular biology, nucleic acidchemistry, and protein chemistry described below are those well knownand commonly employed by those of ordinary skill in the art. Standardtechniques, such as described in Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (supplemented through 1999) (hereinafter“Ausubel”), are used for recombinant nucleic acid methods, nucleic acidsynthesis, cell culture methods, and transgene incorporation, e.g.,electroporation, injection, and lipofection. Generally, oligonucleotidesynthesis and purification steps are performed according tospecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences which are provided throughout this document. The procedurestherein are believed to be well known to those of ordinary skill in theart and are provided for the convenience of the reader.

A variety of additional terms are defined or otherwise characterizedherein.

Polynucleotides of the Invention

Modified Cytokine Nucleic Acids

The invention provides isolated or recombinant modified p40 polypeptidesand modified p35 polypeptides, collectively referred to herein as“modified cytokine polypeptides”, and isolated or recombinantpolynucleotides encoding the polypeptides.

As described in more detail below, in accordance with the presentinvention, polynucleotide sequences which encode modified cytokinepolypeptides, nucleotide sequences (e.g., subsequences) that encodefragments of modified cytokine polypeptides, and nucleotide sequencesthat encode related fusion polypeptides or proteins, or functionalequivalents thereof, are referred to herein as “modified cytokinenucleotides””, “modified p40 nucleotides” or “modified p35 nucleotides,”and more generally as polynucleotides of the invention. Fragments ofeach of the preceding terms are also intended to be included. The term“nucleic acid” is used interchangeably with the term “nucleotide.”

A “corresponding partner polypeptide” or a “corresponding partnersubunit” is defined herein as a polypeptide which associates withanother polypeptide to form a heterodimeric protein (also referred toherein as a “p40/p35” or a “p70” heterodimer) comprising a p40polypeptide and a p35 polypeptide. Thus, a p35 polypeptide is considereda corresponding partner polypeptide to a p40 polypeptide, and a p40polypeptide is considered a corresponding partner polypeptide to a p35polypeptide. Nucleic acids encoding the corresponding partnerpolypeptides (i.e., a p40 polypeptide and a p35 polypeptide) weregenerally co-transfected into mammalian cells to express and secretebiologically active p40/p35 heterodimers into the cell culture media.

In examples provided herein, wild-type p40 nucleic acid (designatedherein as “wt-p40”) comprises the nucleic acid sequence SEQ ID NO:37,encoding a p40 subunit polypeptide comprising the amino acid sequenceSEQ ID NO:15. Likewise, wild-type p35 nucleic acid (designated herein as“wt-p35”) comprises the nucleic acid sequence SEQ ID NO:38, encoding ap35 subunit polypeptide comprising the amino acid sequence SEQ ID NO:36.

Culture supernatants (also referred to herein as “test culturesupernatants”) from cells which comprise a modified p40 nucleic acidand/or a modified p35 nucleic acid of the invention, were generallyassayed in comparison to culture supernatant (also referred to herein as“control culture supernatant”) from cells (also referred to herein as“control cells”) which comprise both wt-p40 and wt-p35 nucleic acids.Such “control cells” express and secrete control wild-type(“wt-p40/wt-p35”) heterodimers.

In some examples provided herein, a nucleic acid of the invention,encoding a polypeptide of the invention, was co-transfected intomammalian cells with a nucleic acid encoding a corresponding wild-type(wt) partner polypeptide (e.g., a modified p40 nucleic acid of theinvention was co-transfected with a wt-p35 nucleic acid, and a modifiedp35 nucleic acid of the invention was co-transfected with a wt-p40nucleic acid). In other examples provided herein, a nucleic acidencoding a polypeptide of the invention was co-transfected intomammalian cells with another nucleic acid of the invention encoding acorresponding modified partner polypeptide (e.g., a modified p40 nucleicacid of the invention was co-transfected with a modified p35 nucleicacid of the invention).

Modified p35 nucleic acids of the invention and the wt-p35 nucleic acidwere in some instances modified to contain a sequence encoding acombined E tag-polyhistidine sequence (“EHtag”) at the extreme 3′ end ofthe coding region (prior to the stop codon). The resulting fusionproteins comprised a p35 polypeptide (e.g., comprising a modified p35polypeptide sequence or a wt-p35 polypeptide sequence) plus an EHtagsequence (AAAGAPVPYPDPLERAAAHHHHHH, identified herein as SEQ ID NO:44)fused to the C-terminus. Control experiments showed no significantdifference in T-cell proliferative activity in the culture media ofcells comprising p35 nucleic acids with and without the EHtagmodification (see, for example, Table 3).

Culture supernatants from mammalian cell transfectants expressing andsecreting heterodimers comprising a modified polypeptide of theinvention and its corresponding wild-type subunit partner, also referredto herein as “(modified/wt) heterodimers”, were initially screened in ahigh-throughput human T-cell based proliferation assay as described inExample 1. Clones corresponding to culture supernatants expressing(modified/wt) heterodimers having a T-cell proliferative activityapproximately equal to or greater than that of a control culturesupernatant expressing a control (wt-p40/wt-p35) heterodimer wereselected for further analysis. The modified p40 nucleic acids andmodified p35 nucleic acids so selected were re-transfected intomammalian cells with the corresponding wild-type (wt) subunit nucleicacid as described above for more detailed activity and expressionanalyses.

Generally, serial dilutions of test culture supernatants containingsecreted heterodimers comprising modified p40 and/or modified p35polypeptides of the invention were assayed for proliferative activity inthe human T-cell based assays as described in Example 1, and compared toactivities obtained from serial dilutions of control culturesupernatants (containing secreted heterodimers comprising wt-p40 andwt-p35 polypeptides).

The “EC50”, which is the effective concentration of heterodimer requiredto achieve 50% of the maximum proliferative activity measured as, e.g.,CPM (counts per minute) of ³H-thymidine incorporated into T-cells, wasestimated from plots of serial dilution versus CPM. First, the serialdilution of control culture supernatant (comprising wt-p40 and wt-p35polypeptides, i.e., wild-type heterodimer) needed to attain 50% of themaximum proliferative activity was determined from the plot of serialdilution versus CPM, where the maximum proliferative activity wasdefined as the maximum amount of ³H-thymidine incorporation (expressedas CPM) at the highest concentrations (lowest dilutions) of controlculture supernatant. The serial dilution of test culture supernatantrequired to attain the same amount of ³H-thymidine incorporation (50% ofthe maximum CPM) was then estimated from the plot. The n-fold differencein dilution of test culture supernatant compared to dilution of controlculture supernatant required to achieve the same amount of ³H-thymidineincorporation (50% of the maximum CPM) provides the “relative EC50”between the test culture supernatant and the control culturesupernatant.

T-cell proliferative activities of test culture supernatants of cellsco-expressing various exemplary modified p40 nucleic acids of theinvention plus wt-p35 nucleic acid, in comparison to culturesupernatants of control cells co-expressing wt-p40 plus wt-p35 nucleicacids, are shown in FIGS. 2-6. The relative T-cell proliferativeactivities, (expressed as relative EC50) of test culture supernatants ascompared to control culture supernatants are presented in Table 1. Thedata show that cells co-expressing a modified p40 nucleic acid of theinvention together with a wt-p35 nucleic acid secrete biologicallyactive protein into the culture media which, on a volume basis, has fromabout 4-fold to as much as 64-fold higher T-cell proliferative activitythan culture media from cells co-expressing wt-p40 plus wt-p35 nucleicacids. TABLE 1 Relative EC50 values for T-cell proliferative activitiesof culture supernatants comprising modified p40/wt-p35 heterodimersRelative p40 subunit p35 subunit EC50 Figure wt-p40 wt-p35   1⁽¹⁾ (SEQID NO: 37) (SEQ ID NO: 38) R1-6-51 wt-p35 ˜4 (SEQ ID NO: 7) A16-94wt-p35  ˜8 to 16 (SEQ ID NO: 6) B8-96 wt-p35 ˜16 to 32 (SEQ ID NO: 2)C2-22 wt-p35 ˜32 to 64 (SEQ ID NO: 1) A3-48 wt-p35 ˜8 (SEQ ID NO: 5)B2-52 wt-p35 ˜4 (SEQ ID NO: 3) B1-81 wt-p35 ˜8 (SEQ ID NO: 4)⁽¹⁾relative EC50 of control (wt-p40/wt-p35) heterodimer culturesupernatant is 1 by definition

Culture supernatants of cells co-transfected with modified p35 nucleicacids of the invention plus wt-p40 nucleic acid also showedsignificantly enhanced T-cell proliferative activities over the control(wt-p40/wt-p35) culture supernatant (Table 2).

Modified p40 nucleic acids and modified p35 nucleic acids were alsoco-transfected into mammalian cells to generate (modified/modified)heterodimers (also referred to herein as “fully modified heterodimers”).Test culture supernatants of cells expressing and secreting fullymodified heterodimers were assayed for proliferative activity in thehuman T-cell based assay in comparison with control supernatants ofcells expressing and secreting wt-p40/wt-p35 heterodimers (Table 2).TABLE 2 Relative EC50 values for T-cell proliferative activities ofculture supernatants comprising fully modified heterodimers Relative p40subunit p35 subunit EC50 wt-p40 wt-p35    1⁽¹⁾ (SEQ ID NO: 37) (SEQ IDNO: 38) wt-p40 R1-4-87  ˜2 (SEQ ID NO: 25) B8-96 wt-p35 ˜16 to 32 (SEQID NO: 2) B8-96 R2-146 ˜32 (SEQ ID NO: 27) B8-96 R2-162 ˜32 (SEQ ID NO:23) C2-22 wt-p35  ˜4 to 64⁽²⁾ (SEQ ID NO: 1) C2-22 R2-42  ˜8 (SEQ ID NO:16) C2-22 R2-157  ˜8 (SEQ ID NO: 24) C2-22 R2-631 ˜64 (SEQ ID NO: 19)C2-22 R2-796 ˜64 (SEQ ID NO: 18) C2-22 R2-555 ˜64 to 128 (SEQ ID NO: 22)C2-22 R2-571 ˜64 to 128 (SEQ ID NO: 21) C2-22 R2-631 ˜64 (SEQ ID NO: 19)C2-22 R2-796 ˜64 (SEQ ID NO: 18)⁽¹⁾Relative EC50 of control (wt-p40/wt-p35) heterodimer culturesupernatant is 1 by definition.⁽²⁾Typically, a 32- to 64-fold enhancement was observed for C2-22/wt-p35heterodimer culture supernatants; the 4-fold enhancement reflects apossibly aberrant result from a single experiment.

Addition of an EHtag sequence to the C-termini of p35 polypeptidesresulted in no significant difference in T-cell proliferative activitiesof the culture supernatants of cells comprising modified p35 nucleicacids with and without the added EHtag (Table 3). Furthermore, additionof four amino acids Leu-Glu-Ser-Ser (LESS in single-letter amino aciddesignation) to the C-terminus of the p35 polypeptide sequence alsoresulted in no significant difference in T-cell proliferative activities(Table 3). TABLE 3 Effect of addition of C-terminal amino acids LESSand/or C-terminal EHtag on relative EC50 values for T-cell proliferativeactivities of culture supernatants Relative p40 subunit p35 subunit EC50wt-p40 wt-p35    1⁽¹⁾ C2-22 R2-555 + LESS⁽²⁾ ˜64-128 C2-22 R2-555 +LESS + EHtag  ˜64 C2-22 R2-555 + EHtag ˜128 C2-22 R2-571 + LESS ˜64-128C2-22 R2-571 + LESS + EHtag ˜128 C2-22 R2-571 + EHtag ˜128⁽¹⁾Relative EC50 of control(wt-p40/wt-p35) heterodimer culturesupernatant is 1 by definition.⁽²⁾“+LESS” indicates addition of the amino acids Leu-Glu-Ser-Ser, and“+EHtag” indicates addition of EHtag sequence to the C-terminus of thep35 polypeptide sequence, in the order indicated

The relative amounts of modified heterodimeric protein and wild-typeheterodimeric protein secreted into the cell culture media wasquantitated by Western blotting of culture supernatant dilutions asdescribed in Example 1. FIG. 7 shows that a significantly larger amountof p40/p35 heterodimer (labeled “p70” in the Figure) is consistentlyproduced by cells expressing modified p40 plus wt-p35 nucleic acids ascompared to control cells expressing wt-p40 plus wt-p35 nucleic acids.Thus, modified nucleic acids of the invention appear to promote enhancedproduction (i.e., expression and/or secretion) of modified heterodimersas compared to wild-type (wt-p40/wt-p35) heterodimers.

As noted above and shown in FIG. 3, culture media from cells expressingand secreting the modified C2-22/wt-p35 heterodimer showed on averagethe highest overall proliferative activity, with an up to 64-foldincrease in relative proliferative activity over that of control culturesupernatant from cells expressing and secreting the wild-type(wt-p40/wt-p35) heterodimer (that is, up to a 64-fold lower volume ofculture supernatant from cells expressing the modified heterodimer wasrequired to achieve an EC50 activity level equivalent to that of a 1×volume of culture media from cells expressing the wild-type(wt-p40/wt-p35) heterodimer).

To estimate the relative contributions of heterodimer production andactivity to the overall proliferative activity observed in culturesupernatants, heterodimers were first quantitated by immunoblot, usingan anti-p35 monoclonal antibody (mAB), of equivalent dilutions ofpurified heterodimer from C2-22/wt-p35 and wt-p40/wt-p35 cultures. Fromdensitometery analysis, an estimated 16-fold enhancement of expressionof C2-22/wt-p35 heterodimer over that of the control wt-p40/wt-p35heterodimer was observed (FIG. 8).

Based on this quantitation, purified C2-22/wt-p35 heterodimer exhibitedabout a four-fold higher proliferative activity compared to anequivalent concentration of purified control wt-p40/wt-p35 heterodimerin the human T-cell proliferation assay, after normalizing for proteinconcentration (FIG. 9). This result suggests that the up to 64-foldenhancement of proliferative activity in culture supernatants of cellsexpressing C2-22/wt-p35 heterodimers as compared to wild-typeheterodimer is consistent with about a 16-fold greater concentration ofactive heterodimer produced, together with about a four-fold higherproliferative activity of the isolated C2-22/wt-p35 heterodimer overthat of the isolated control wild-type heterodimer.

The ability of purified heterodimers comprising modified p35 and/ormodified p40 polypeptides of the invention to induce production of theT_(H)1-specific cytokine interferon-γ in human T-cells was determined inthe human T_(H)1-differentiation/IFN-γ induction assay. FIG. 10 showsthe concentrations of human IFN-γ produced by human T-cells incubated inthe presence or absence of purified C2-22/wt-p35 heterodimer, purifiedwt-p40/wt-p35 heterodimer, or a commercially available purifiedinterleukin-12 cytokine standard, followed by activation with anti-CD3and anti-CD28 antibodies. The concentration of IFN-γ produced by humanT-cells incubated with ˜1 nanogram/milliliter (ng/ml) of partiallypurified p40C2-22/wt-p35 heterodimer was significantly greater than thatof cells treated with ˜1 ng/ml of partially purified controlwt-p40/wt-p35 heterodimer.

A fully modified heterodimer of the invention, designatedC2-22/R2-571-EHtag, comprising a modified p40 polypeptide of theinvention comprising a sequence identified as the mature polypeptideregion (amino acid residues 23-324) of SEQ ID NO:8, plus a modified p35polypeptide of the invention comprising a sequence identified as themature polypeptide region (amino acid residues 23-219) of SEQ ID NO:31,plus the C-terminal EHtag, was purified and quantitated by absorbance at280 nanometers (nm) as described in Example 1. FIG. 12 shows that theT-cell proliferative activity of the purified fully modified heterodimeris about 8 times greater than that of purified wild-type(wt-p40/wt-p35-EHtag) heterodimer, after normalizing for proteinconcentration. FIG. 13 shows that the purified fully modifiedheterodimer induced a concentration level of IFN-γ production in humanT-cells than that of the purified wild-type heterodimer. Because themaximum concentration of IFN-γ production could not be derived from thedata, an EC50 value could not be estimated for either the purified fullymodified or wild-type heterodimer preparations. However, FIG. 13 showsthat a concentration of ˜10 ng/ml of purified, fully modifiedheterodimer induces a similar concentration of IFN-γ production in theT-cell based assay as ˜50 ng/ml of purified, wild-type heterodimer,suggesting that the fully modified heterodimer exhibits an estimatedthree-fold to five-fold greater IFN-γ induction activity than thewild-type heterodimer.

Co-expressing in mammalian cells a modified p40 nucleic acid of theinvention plus a modified p35 nucleic acid of the invention produces asubstantially more homogenous heterodimeric protein than theheterodimeric protein produced by co-expressing a wt-p40 nucleic acidand a wt-p35 nucleic acid. FIG. 14 shows a Western blot, probed using ananti-E tag mAB (Pharmacia-Amersham), of purified heterodimers producedby co-expressing a modified p40 nucleic acid of the invention(comprising the C2-22 polynucleotide sequence SEQ ID NO:1) plus amodified p35 nucleic acid of the invention (comprising the R2-571polynucleotide sequence SEQ ID NO:21), and purified heterodimersproduced by co-expressing wt-p40 and wt-p35 nucleic acids (comprisingthe polynucleotide sequences SEQ ID NO:37 and SEQ ID NO:38,respectively). Both the modified p35 nucleic acid and the wt-p35 nucleicacid also comprised a C-terminal EHtag coding sequence. As seen in theFigure, co-expression of the wt-p40 and wt-p35 nucleic acids producedprotein which electrophoresed in multiple bands with apparent molecularweights (MW) of ˜70 kDa or greater, while co-expression of the exemplarymodified p40 and modified p35 nucleic acids produced protein whichelectrophoresed as essentially a single band with an apparent molecularweight of approximately 70 kDa.

Without being limited by theory, the observed enhancement of production(i.e., expression and/or secretion) of modified heterodimers from cellsexpressing nucleic acids of the invention over that of cells expressingwt-p40 plus wt-p35 nucleic acids may be attributable to one or more of anumber of factors, including, but not limited to, e.g., increasedexpression (e.g., increased mRNA production), more efficient secretionof expressed polypeptide, more optimal folding of expressed polypeptide,more efficient processing of expressed polypeptide to maturepolypeptide, enhanced dimerization affinity of expressed polypeptides,etc. For example, modified p40 and/or modified p35 polypeptides of theinvention may exhibit enhanced dimerization affinity with thecorresponding subunit polypeptide, resulting, for example, from anincreased amount of one or both polypeptide subunits in the culturemedia (thus shifting monomer-heterodimer equilibrium towards heterodimerformation in solution) and/or from more favorable protein-proteininteractions at the subunit interfaces. Such enhanced dimerizationaffinity may be manifested, for example, in a more favorable subunitassociation constant (K_(assoc)), and/or a decreased dissociation rateconstant (k_(off)). Enhanced dimerization affinity may shift themonomer-heterodimer equilibrium towards heterodimer, resulting in theincreased production of biologically active molecules. Furthermore, fromFIG. 14, it is apparent that expression of nucleic acids of theinvention results in the production of a highly homogeneous biologicallyactive heterodimeric protein.

An isolated heterodimer comprising a modified p40 polypeptide and/or amodified p35 polypeptide of the invention thus exhibits proliferativeactivity and/or IFN-γ induction activity in human T-cells. In variousembodiments, an isolated heterodimer comprising a modified p40polypeptide and/or a modified p35 polypeptide of the invention has atleast about 1.5-fold, 2-fold, 4-fold or 8-fold greater T-cellproliferative activity than an isolated heterodimer comprising a wt-p40polypeptide and a wt-p35 polypeptide, wherein the wt-p40 polypeptidecomprises the mature polypeptide sequence identified as amino acidresidues 23 to 328 of SEQ ID NO:15 and the wt-p35 polypeptide comprisesthe mature polypeptide sequence identified as amino acid residues 23 to219 of SEQ ID NO:36.

Modified p40 nucleic acids and modified p35 nucleic acids of theinvention, compared to wt-p40 and wt-p35 nucleic acids, show enhancedproduction of highly homogenous, biologically active heterodimericcytokine molecules when expressed in mammalian cells. This featurerenders the modified p40 nucleic acids and the modified p35 nucleicacids of the invention particularly useful in, e.g., gene therapy orother applications where genetic delivery of localized amounts of highlyactive molecules is an important consideration.

Modified p40 Nucleic Acids

Exemplary nucleic acids which encode modified p40 polypeptides of theinvention having proliferative activity and/or IFN-γ induction activityin a T-cell based assay (such as, e.g., a human T-cell based assay) areidentified herein as SEQ ID NO:1 to SEQ ID NO:7, encoding modified p40polypeptides identified herein as SEQ ID NO:8 to SEQ ID NO:14,respectively. These nucleic acids comprise the following maturepolypeptide coding regions: nucleotides 67 to 972 of SEQ ID NO:1;nucleotides 67 to 981 of SEQ ID NO:2; nucleotides 67 to 981 of SEQ IDNO:3; nucleotides 67 to 981 of SEQ ID NO:4, nucleotides 67 to 966 of SEQID NO:5; nucleotides 67 to 972 of SEQ ID NO:6; and nucleotides 67 to 987of SEQ ID NO:7, which encode the mature polypeptide regions identifiedas: ammo acid residues 23-324 of SEQ ID NO:8; amino acid residues 23-327of SEQ ID NO:9, amino acid residues 23-327 of SEQ ID NO:10, amino acidresidues 23-327 of SEQ ID NO:11, amino acid residues 23-322 of SEQ IDNO:12, amino acid residues 23-324 of SEQ ID NO:13, and amino acidresidues 23-329 of SEQ ID NO:14. These nucleic acids also comprise thefollowing leader peptide coding regions: nucleotides 1 to 66 of SEQ IDNO:1-SEQ ID. NO:7, which encode the leader peptide regions identified asamino acid residues 1-22 of SEQ ID NOS:8-14.

In one aspect, the invention provides an isolated or recombinant nucleicacid that comprises a polynucleotide sequence selected from the groupof: (a) the mature polypeptide coding region of SEQ ID NO:1 to SEQ IDNO:7, or a complementary polynucleotide sequence thereof; (b) apolynucleotide sequence encoding a polypeptide sequence selected fromthe mature polypeptide region of SEQ ID NO:8 to SEQ ID NO:14, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which hybridizes under at least stringent or at least highlystringent hybridization conditions (or ultra-high stringent orultra-ultra-high stringent hybridization conditions) over substantiallythe entire length of polynucleotide sequence (a) or (b), or with a 30,50, 100, 200, 300, 400, 500, 600, 700, 800, 850, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, or 980 nucleotide base subsequence orfragment of a polynucleotide sequence of (a) or (b); (d) apolynucleotide sequence which encodes a polypeptide comprising an aminoacid sequence having at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99%amino acid sequence identity to a mature polypeptide region of asequence selected from SEQ ID NO:8 to SEQ ID NO:14; and (e) apolynucleotide sequence comprising a fragment of (a), (b), (c), or (d),which fragment encodes all or a part of a polypeptide havingproliferative activity or interferon-gamma induction activity in aT-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or amodified p35 polypeptide).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p40 polypeptidesequence, the modified p40 polypeptide sequence comprising an amino acidmodification located at an amino acid position equivalent to (i.e., an“equivalent position” to) that in the amino acid sequence of a wild-typep40 polypeptide (SEQ ID NO:15). The modification can include: (a) asubstitution of the specified amino acid for a different amino acid atone or more position, equivalent to that of SEQ ID NO:15, selected fromLeu62, Ser71, Gln78, His99, Thr127, Arg130, Lys185, Glu186, Tyr187,Glu188, Ser190, Asp196, Met211, Val289, Ser305, Ser307, Arg309, andGln311; (b) a deletion of one or more amino acid residues at equivalentposition Arg181 to Asn184 inclusive or a substitution of the amino acidresidues at equivalent positions Arg181 to Asn184 inclusive for theamino acid residues Ser-(Leu or Met)-(Glu or Asp)-His-Arg; (c) adeletion of one or more amino acid at equivalent positions Asp287 andArg288. The modified p40 polypeptide may optionally include two or moreof modification (a), (b) or (c). The modified p40 polypeptide sequenceencoded by the nucleic acid of the invention may be a modified sequenceof a naturally-occurring or wild-type p40 polypeptide sequence of amammal (e.g., human, primate, ruminant, or rodent), preferably primate,more preferably human. Preferred substitutions include Leu62Ser;Ser71Thr; Gln78His; His99(Arg or Gln); Thr127(Ser or Ile); Arg130Lys;Lys185Glu; Glu186Tyr; Tyr187(Lys or Asn); Glu188Lys; Ser190(Arg or Thr);Asp196Gly; Met211Val; Val289(Ile or Leu); Ser305Lys; Ser307Arg;Arg309Gln; and Gln311Arg. The invention also includes a polynucleotidesequence encoding said polypeptide or a fragment of said polypeptidehaving proliferative activity or IFN-γ induction activity in a T-cellbased assay (such as, e.g., a human T-cell based assay), in the presenceof a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or a modifiedp35 polypeptide).

The modified p40 polypeptide sequence encoded by a nucleic acid of theinvention optionally comprises an amino acid modification, located at anamino acid residue position equivalent to that in the amino acidsequence of a p40 polypeptide (SEQ ID NO:15), selected from: Cys2His;His3Pro; Ile8Val; Phe15Leu; Val21Met; Lys27Glu; Asp29Asn; Asp40Asn;Met45Thr; Thr49Ala; Glu67Gly, His91Arg; Glu95(Ala or Thr), Val96Ala;Glu122Lys; Asn125Ala; Asn135Asp; Arg139His; Thr147Ala; Thr153Lys;Ser155Thr; Ser163Thr; Gln166(Arg or His), Ala172Thr; Ala173Val;Thr174Leu; Ala177Glu; Glu178Asp; Arg179Leu; Val180Gly; Ala201Ser;Val212Leu; Asp213Glu; Val215Ile; Ser226Arg; Lys244Arg; Gln251His;Val254Ile; Ser255Asn; Glu257Gly; Thr264(Ala or Ile); Thr272Met;Cys274Gly; Val275Ile; Lys280Arg; Ser281Asn; Lys285Asp; Lys286Arg;Phe290Ser; Thr291(Met or Val); Lys293Gln; Thr297Lys; Ile299(Thr or Val);Arg301His; Asn303Asp; Ser318Phe; Glu321Asp; Pro326Ser; Cys327Leu; andSer328(Gly or Gln).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p40polypeptide, wherein the modified p40 polypeptide comprises an aminoacid sequence having at least about 90% amino acid sequence identity tothe amino acid sequence identified herein as the mature polypeptideregion (amino acid residue positions 23-328) of SEQ ID NO:39:IWEL-X₂₇-K-X₂₉-VYVVELDWYP-X₄₀-APGE-X₄₅-VVL-X₄₉-CDTPEEDGITWT-X₆₂-DQSS-X₆₇-VLG-X₇₁-GKTLTI-X₇₈-VKEFGDAGQYTC-X₉₁-KGG-X₉₅-X₉₆-LS-X₉₉-SLLLLHKKEDGIWSTDILKDQK-X₁₂₂-PK-X₁₂₅-K-X₁₂₇-FL-X₁₃₀-CEAK-X₁₃₅-YSG-X₁₃₉-FTCWWLT-X₁₄₇-ISTDL-X₁₅₃-F-X₁₅₅-VKSSRGS-X₁₆₃-DP-X₁₆₆-GVTCG-X₁₇₂-X₁₇₃-X₁₇₄-LS-X₁₇₇-X₁₇₈-X₁₇₉-X₁₈₀-X₁₈₁-X₁₈₂-X₁₈₃-X₁₈₄-X₁₈₅-X₁₈₆-X₁₈₇-X₁₈₈-Y-X₁₉₀-VECQE-X₁₉₆-SACP-X₂₀₁-AEESLPIEV-X₂₁₁-X₂₁₂-X₂₁₃-A-X₂₁₅-HKLKYENYTS-X₂₂₆-FFIRDIIKPDPPKNLQL-X₂₄₄-PLKNSR-X₂₅₁-VE-X₂₅₄-X₂₅₅-W-X₂₅₇-YPDTWS-X₂₆₄-PHSYFSLTF-X₂₇₄-X₂₇₅-QVQG-X₂₈₀-X₂₈₁-KRE-X₂₈₅-X₂₈₆-X₂₈₇-X₂₈₈-X₂₈₉-F-X₂₉₁-D-X₂₉₃-TSA-X₂₉₇-V-X₂₉₉-C-X₃₀₁-K-X₃₀₃-A-X₃₀₅-I-X₃₀₇-V-X₃₀₉-A-X₃₁₁-DRY-X₃₁₅-SS-X₃₁₈-WS-X₃₂₁-WASV-X₃₂₆-X₃₂₇-X₃₂₈,or a conservatively substituted variation thereof, where X₂₇ is K or E;X₂₉ is D or N; X₄₀ is D or N; X₄₅ is M or T; X₄₉ is T or A; X₆₂ is S;X₆₇ is E or G; X₇₁ is T; X₇₈ is H; X₉₁ is H or R; X₉₅ is E, A, K, or T,X₉₆ is V or A; X₉₉ is R or Q; X₁₂₂ is E or K; X₁₂₅ is N or A; X₁₂₇ is Sor I; X₁₃₀ is K; X₁₃₅ is N or D; X₁₃₉ is R or H; X₁₄₇ is T or A; X₁₅₃ isT or K; X₁₅₅ is S or T; X₁₆₃ is S or T; X₁₆₆ is Q, R, or H; X₁₇₂ is A orT; X₁₇₃ is A or V; X₁₇₄ is T or L; X₁₇₇ is A or E; X₁₇₈ is E or D; X₁₇₉is R, L, or K; X₁₈₀ is V or G; X₁₈₁ to X₁₈₄ inclusive is deleted, or isreplaced with the sequence S-(L or M)-(E or D)-H-R; X₁₈₅ is E; X₁₈₆ isY; X₁₈₇ is K or N; X₁₈₈ is K; X₁₉₀ is R or T; X₁₉₆ is G; X₂₀₁ is A or S;X₂₁₁ is V; X₂₁₂ is V or L; X₂₁₃ is D or E; X₂₁₅ is V or I; X₂₂₆ is S orR; X₂₄₄ is K or R; X₂₅₁ is Q or H; X₂₅₄ is V or I; X₂₅₅ is S or N; X₂₅₇is E or G; X₂₆₄ is T or A; X₂₇₄ is C or G; X₂₇₅ is V or I; X₂₈₀ is K orR; X₂₈₁ is S or N; X₂₈₅ is K or D; X₂₈₆ is K or R; X₂₈₇ is D or isdeleted; X₂₈₈ is R or is deleted; X₂₈₉ is I or L; X₂₉₁ is T or M; X₂₉₃is K or Q; X₂₉₇ is T or K; X₂₉₉ is I, T, or V; X₃₀₁ is R or H; X₃₀₃ is Nor D; X₃₀₅ is K; X₃₀₇ is R; X₃₀₉ is Q; X₃₁₁ is R; X₃₁₅ is Y or H; X₃₁₈is S or F; X₃₂₁ is E or D; X₃₂₆ is P or S; X₃₂₇ is C or L; and X₃₂₈ isS, G, or Q. In various embodiments, the modified p40 polypeptide encodedby the nucleic acid of the invention comprises an amino acid sequencehaving at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to the mature polypeptide region (amino acid residuepositions 23-328) of SEQ ID NO:39. The invention also includes apolynucleotide sequence encoding said polypeptide or a fragment of saidpolypeptide having proliferative activity or IFN-γ induction activity ina T-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or amodified p35 polypeptide).

The modified p40 polypeptide encoded by a nucleic acid of the inventionmay further comprise a leader peptide sequence having at least about90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to theamino acid sequence M-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identifiedherein as the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:39, where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅ isF or L; and X₂₁ is V or M.

The present invention also includes a modified p40 polypeptidecomprising a conservatively modified variation of the amino acidsequence identified herein as the mature polypeptide region (amino acidresidue positions 23-328) of SEQ ID NO:39, and, optionally, aconservatively modified variation of the leader peptide region (aminoacid residue positions 1-22) of SEQ ID NO:39 and a polynucleotidesequence encoding said polypeptide or a fragment of said polypeptidehaving proliferative activity or IFN-γ induction activity in a T-cellbased assay (such as, e.g., a human T-cell based assay), in the presenceof a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or a modifiedp35 polypeptide).

The invention also includes a modified p40 polypeptide comprising theamino acid sequence identified herein as the mature polypeptide region(amino acid residue positions 23-328) of SEQ ID NO:39, and, optionally,comprising the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:39, and a polynucleotide sequence encoding said polypeptideor a fragment of said polypeptide having proliferative activity or IFN-γinduction activity in a T-cell based assay (such as, e.g., a humanT-cell based assay), in the presence of a p35 polypeptide (such as,e.g., a wt-p35 polypeptide or a modified p35 polypeptide).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a polypeptide comprising aleader peptide sequence having at least about 90% amino acid sequenceidentity to the amino acid sequenceM-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identified herein as the leaderpeptide region (amino acid residue positions 1-22) of SEQ ID NO:39,where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅ is F or L; and X₂₁is V or M. In various embodiments, the leader peptide sequence encodedby the nucleic acid of the invention comprises an amino acid sequencehaving at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to leader peptide region (amino acid residues 1-22) ofSEQ ID NO:39. In other embodiments, the leader peptide sequencecomprises the sequence identified as the leader peptide region (aminoacid residue positions 1-22) of SEQ ID NO:39, or conservatively modifiedvariations thereof.

Modified p35 Nucleic Acids

Exemplary nucleic acids which encode modified p35 polypeptides havingproliferative activity and/or IFN-γ induction activity in theT-cell-based assay are identified herein as SEQ ID NO:16 to SEQ IDNO:25, encoding modified p35 polypeptides identified herein as SEQ IDNO:26 to SEQ ID NO:35, respectively. These nucleic acids comprise thefollowing mature polypeptide coding regions: nucleotides 76 to 663 ofSEQ ID NO:16; nucleotides 76 to 663 of SEQ ID NO:17; nucleotides 67 to657 of SEQ ID NO: 18; nucleotides 67 to 657 of SEQ ID NO:19, nucleotides67 to 657 of SEQ ID NO:20; nucleotides 67 to 657 of SEQ ID NO:21;nucleotides 67 to 657 of SEQ ID NO:22; nucleotides 67 to 657 of SEQ IDNO:23; nucleotides 67 to 657 of SEQ ID NO:24; and nucleotides 67 to 657of SEQ ID NO:25, which encode the mature polypeptide regions identifiedas: amino acid residues 26-221 of SEQ ID NO:26, amino acid residues26-221 of SEQ ID NO:27, amino acid residues 23-219 of SEQ ID NO:28,amino acid residues 23-219 of SEQ ID NO:29, amino acid residues 23-219of SEQ ID NO:30, amino acid residues 23-219 of SEQ ID NO:31, amino acidresidues 23-219 of SEQ ID NO:32, amino acid residues 23-219 of SEQ IDNO:33, amino acid residues 23-219 of SEQ ID NO:34, and amino acidresidues 23-219 of SEQ ID NO:35. These nucleic acids also comprise thefollowing leader peptide coding regions: nucleotides 1 to 75 of SEQ IDNO:116-SEQ ID NO:17, and nucleotides 1 to 66 of SEQ ID NO:18-SEQ IDNO:25, which encode the leader peptide regions identified as: amino acidresidues 1-25 of SEQ ID NOS:26 to 27, and amino acid residues 1-22 ofSEQ ID NOS:28 to 35.

In one aspect, the invention provides an isolated or recombinant nucleicacid that comprises a polynucleotide sequence selected from the groupof: (a) the mature polypeptide coding region of SEQ ID NO:16 to SEQ IDNO:35, or a complementary polynucleotide sequence thereof; (b) apolynucleotide sequence encoding a polypeptide sequence selected fromthe mature polypeptide region of SEQ ID NO:26 to SEQ ID NO:35, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which hybridizes under at least stringent or at least highlystringent hybridization conditions (or ultra-high stringent orultra-ultra-high stringent hybridization conditions) over substantiallythe entire length of polynucleotide sequence (a) or (b), or with a 30,50, 100, 200, 300, 400, 500, 550, 580, 590, 600, 610, 620, 630, 640,650, 660, or 670 nucleotide base subsequence or fragment of apolynucleotide sequence of (a) or (b); (d) a polynucleotide sequencewhich encodes a polypeptide comprising an amino acid sequence having atleast about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentity to a mature polypeptide region of a sequence selected from SEQID NO:26 to SEQ ID NO:35; and (e) a polynucleotide sequence comprising afragment of (a), (b), (c), or (d), which fragment encodes all or a partof a polypeptide having proliferative activity or IFN-γ inductionactivity in a T-cell based assay (such as, e.g., a human T-cell basedassay), in the presence of a p40 polypeptide (such as, e.g., a wt-p40polypeptide or a modified p40 polypeptide).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a modified p35 polypeptidecomprising an amino acid modification located at an amino acid residueposition equivalent to (i.e., an equivalent position to) that in theamino acid sequence of a wild-type p35 polypeptide (SEQ ID NO:36). Themodification can include: (a) a substitution of the specified amino acidfor a different amino acid at one or more equivalent position to that ofSEQ ID NO:36 selected from Thr91, Met120, Ala121, Val212, Thr213, andAla218; (b) a insertion of one or more amino acid residues Phe-His-Leubetween equivalent positions Leu19 and Ser20; (c) a deletion of theamino acid at equivalent position Pro36. The modified p35 polypeptidemay optionally include two or more of modification (a), (b), or (c). Themodified p35 polypeptide sequence encoded by the nucleic acid of theinvention may be a modified sequence of a naturally-occurring orwild-type p35 polypeptide sequence of a mammal (e.g., human, primate,ruminant, or rodent), preferably primate, more preferably human.Preferred substitutions include Thr91(Ala or Ile); Met120Thr; Ala121Thr;Val212Met; Thr213Met; and Ala218Ser. The invention also includes apolynucleotide sequence encoding said polypeptide or a fragment of saidpolypeptide having proliferative activity or IFN-γ induction activity ina T-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p40 polypeptide (such as, e.g., a wt-p40 polypeptide or amodified p40 polypeptide).

The modified p35 polypeptide sequence encoded by a nucleic acid of theinvention optionally comprises an amino acid modification, located at anamino acid position equivalent to that in the amino acid sequence of ap35 polypeptide (SEQ ID NO:36), selected from: Cys2Tyr; Ala4(Leu orPro); Ser6Gly; Val10Ile; Ala11Ser; Asp17His; Ala22Gly; Asn24Ser;Val27Thr; Ala28Thr; Pro30Ala; Asp31(Ser or Gly); Met34Arg; Phe35(Ser orLeu); Pro36(deleted); His39Asp; His40Tyr; Arg46Lys; Val48Ala; Met51Thr;Lys54Arg; Thr58Ile; Pro63Ser; Ile69Thr; Lys76Gln; Lys92Thr; Asn98Ala;Glu101Gly; Thr102Ile; Phe104Leu; Leu124His; Ser125Gly; Val136Met;Thr140Ala; Asp148Asn; Ala161Thr; Val162Ala; Asp164Ala; Met167Leu;Phe172Val; Val177Ala; Ser181Pro; Pro186Leu; Asp210Asn; and insertion ofone or more of 220Leu; 221 Glu; 222Ser; and 223 Ser.

The invention also includes an isolated or recombinant nucleic acidcomprising a polynucleotide sequence encoding a modified p35polypeptide, wherein the modified p35 polypeptide comprises an aminoacid sequence having at least about 90% amino acid sequence identity tothe amino acid sequence identified herein as the mature polypeptideregion (amino acid residue positions 23-219) of SEQ ID NO:40:R-X₂₄-LP-X₂₇-X₂₈-T-X₃₀-X₃₁-PG-X₃₄-X₃₅-X₃₆-CL-X₃₉-X₄₀-SQNLL-X₄₆-A-X₄₈-SN-X₅₁-LQ-X₅₄-A-X₅₆-Q-X₅₈-LEFY-X₆₃-CTSEE-X₆₉-DHEDIT-X₇₆-DKTSTVEACLPLEL-X₉₁-X₉₂-NESCL-X₉₈-SR-X₁₀₁-X₁₀₂-S-X₁₀₄-ITNGSCLASRKTSFM-X₁₂₀-X₁₂₁-LC-X₁₂₄-X₁₂₅-SIYEDLKMYQ-X₁₃₆-EFK-X₁₄₀-MNAKLLM-X₁₄₈-PKRQIFLDQNML-X₁₆₁-X₁₆₂-I-X₁₆₄-EL-X₁₆₇-QALN-X₁₇₂-NSET-X₁₇₇-PQK-X₁₈₁-SLEE-X₁₈₆-DFYKTKIKLCILLHAFRIRAVTI-X₂₁₀-R-X₂₁₂-X₂₁₃-SYLN-X₂₁₈-S,or a conservatively substituted variation thereof, where X₂₄ is N or S;X₂₇ is V or T; X₂₈ is A or T; X₃₀ is P or A; X₃₁ is D, S, or G; X₃₄ is Mor R; X₃₅ is F, S, or L; X₃₆ is P or is deleted; X₃₉ is H or D; X₄₀ is Hor Y; X₄₆ is R or K; X₄₈ is V or A; X₅₁ is M or T; X₅₄ is K or R; X₅₆ isK or R; X₅₈ is T or I; X₆₃ is P or S; X₆₉ is I or T; X₇₆ is K or Q; X₉₁is A or I; X₉₂ is K or T; X₉₈ is N or A; X₁₀₁ is E or G; X₁₀₂ is T or I;X₁₀₄ is F or L; X₁₂₀ is T; X₁₂₁ is T; X₁₂₄ is L or H; X₁₂₅ is S or G;X₁₃₆ is V or M; X₁₄₀ is T or A; X₁₄₈ is D or N; X₁₆₁ is A or T; X₁₆₂ isV or A; X₁₆₄ is D or A; X₁₆₇ is M or L; X₁₇₂ is F or V; X₁₇₇ is V or A;X₁₈₁ is S or P; X₁₈₆ is P or L; X₂₁₀ is D or N; X₂₁₂ is M; X₂₁₃ is M;and X₂₁₈ is S. In various embodiments, the nucleic acid of the inventionencodes a modified p35 polypeptide comprising an amino acid sequencehaving at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to the mature polypeptide region (amino acid residuepositions 23-219) of SEQ ID NO:40. The invention also includes apolynucleotide sequence encoding said polypeptide or a fragment of saidpolypeptide having proliferative activity or IFN-γ induction activity ina T-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p40 polypeptide (such as, e.g., a wt-p40 polypeptide or amodified p40 polypeptide).

The modified p35 polypeptide encoded by a nucleic acid of the inventionmay further comprise a leader peptide sequence having at least about90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to theamino acid sequence M-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₇-HLSL-X₂₂,identified herein as the leader peptide region (amino acid residuepositions 1-22) of SEQ ID NO:40, where X₂ is C or Y; X₄ is A, L or P; X₆is S or G; X₁₀ is V or I; X₁₁ is A or S; X₁₇ is D or H; and X₂₂ is A orG, and optionally includes an insertion of the amino acids P-H-L betweenpositions 18 and 19.

The present invention also includes a modified p35 polypeptidecomprising a conservatively modified variation of the amino acidsequence identified herein as the mature polypeptide region (amino acidresidue positions 23-219) of SEQ ID NO:40, and, optionally, aconservatively modified variation of the leader peptide region (aminoacid residue positions 1-22) of SEQ ID. NO:40, and a polynucleotidesequence encoding said polypeptide or a fragment of said polypeptidehaving proliferative activity or IFN-γ induction activity in a T-cellbased assay (such as, e.g., a human T-cell based assay), in the presenceof a p40 polypeptide (such as, e.g., a wt-p40 polypeptide or a modifiedp40 polypeptide).

The invention also includes a modified p35 polypeptide comprising theamino acid sequence identified herein as the mature polypeptide region(amino acid residue positions 23-219) of SEQ ID NO:40, and, optionally,comprising the leader peptide region. (amino acid residue positions1-22) of SEQ ID NO:40, and a polynucleotide sequence encoding saidpolypeptide or a fragment of said polypeptide having proliferativeactivity or IFN-γ induction activity in a T-cell based assay (such as,e.g., a human T-cell based assay), in the presence of a p40 polypeptide(such as, e.g., a wt-p40 polypeptide or a modified p40 polypeptide).

The invention also includes an isolated or recombinant nucleic acid,comprising a polynucleotide sequence encoding a polypeptide comprising aleader peptide sequence having at least about 90% amino acid sequenceidentity to the amino acid sequenceM-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₆-HLSL-X₂₂, identified herein as theleader peptide region (amino acid residue positions 1-22) of SEQ IDNO:40, where X₂ is C or Y; X₄ is A, L, or P; X₆ is S or G; X₁₀ is V orI; X₁₁ is A or S; X₁₆ is D or H; X₂₂ is A or G, and optionally includesan insertion of the amino acids P-H-L between positions 18 and 19. Invarious embodiments, the leader peptide sequence encoded by the nucleicacid of the invention comprises an amino acid sequence having at leastabout 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identityto leader peptide region (amino acid residue positions 1-22) of SEQ IDNO:40. In another embodiment, the leader peptide sequence comprises thesequence identified as the leader peptide region (amino acid residuepositions 1-22) of SEQ ID NO:40, or conservatively modified variationsthereof.

As described in greater detail below, the polynucleotides of theinvention are useful in a variety of applications, including, but notlimited to, as therapeutic and prophylactic agents in methods of in vivoand ex vivo treatment of a variety of diseases, disorders, andconditions in a variety of subjects; for use in in vitro methods, suchas diagnostic methods, to detect, diagnose, and treat a variety ofdiseases, disorders, and conditions in a variety of subjects; for usein, e.g., gene therapy; as therapeutics and prophylactics, e.g., for usein methods of therapeutic and prophylactic treatment of a disease,disorder or condition; as immunogens; and for use in diagnostic andscreening assays; and as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of nucleic acids encoding modified p40 polypeptides andmodified p35 polypeptides).

Making Polynucleotides of the Invention

Polynucleotides and oligonucleotides of the invention can be prepared bystandard solid-phase methods, according to known synthetic methods.Typically, fragments of up to about 20, 30, 40, 50, 60, 70, 80, 90,and/or 100 nucleotide bases are individually synthesized, then joined(e.g., by enzymatic or chemical ligation methods, or polymerase mediatedrecombination methods) to form essentially any desired continuoussequence. In another aspect, nucleotide fragments of greater than 100nucleotide bases (e.g., 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, or 950 bases) are individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated recombination methods) to formessentially any desired continuous sequence. For example, thepolynucleotides and oligonucleotides of the invention, includingfragments thereof (and those as described herein), can be prepared bychemical synthesis using, e.g., the classical phosphoramidite methoddescribed by Beaucage et al. (1981) Tetrahedron Letters 22:1859-69, orthe method described by Matthes et al. (1984) EMBO J. 3:801-05, e.g., asis typically practiced in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company (mcrc@oligos.com), The Great American Gene Company(http://www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.) and many others. Similarly, peptidesand antibodies can be custom ordered from any of a variety of sources,such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc.(http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis,Inc., and many others.

Certain polynucleotides of the invention may also obtained by screeningcDNA libraries (e.g., libraries generated by recombining homologousnucleic acids as in typical diversity generation methods, such as, e.g.,shuffling methods) using oligonucleotide probes which can hybridize toor PCR-amplify polynucleotides which encode the modified p40 or modifiedp35 polypeptides and fragments of those polypeptides. Procedures forscreening and isolating cDNA clones are well-known to those of skill inthe art. Such techniques are described in, for example, Sambrook et al.,Molecular Cloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (hereinafter“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (supplemented through 1999)(hereinafter “Ausubel”).

As described in more detail herein, the polynucleotides of the inventioninclude sequences which encode novel modified p40 polypeptides andmodified p35 polypeptides, and sequences complementary to the codingsequences, and novel fragments of such coding sequences and complementsthereof. The polynucleotides can be in the form of RNA or in the form ofDNA, and include mRNA, cRNA, synthetic RNA and DNA, and cDNA. Thepolynucleotides can be double-stranded or single-stranded, and ifsingle-stranded, can be the coding strand or the non-coding (anti-sense,complementary) strand. The polynucleotides optionally include the codingsequence of an modified 40 polypeptide or a modified p35 polypeptide (i)in isolation, (ii) in combination with additional coding sequence, so asto encode, e.g., a fusion protein, a pre-protein, a prepro-protein, orthe like, (iii) in combination with non-coding sequences, such asintrons, control elements such as a promoter, a terminator element, or5′ and/or 3′ untranslated regions effective for expression of the codingsequence in a suitable host, and/or (iv) in a vector or host environmentin which the modified p40 polypeptide or modified p35 polypeptide codingsequence is a heterologous nucleic acid sequence or gene. Sequences canalso be found in combination with typical compositional formulations ofnucleic acids, including in the presence of carriers, buffers,adjuvants, excipients and the like.

The term DNA or RNA encoding the respective modified p40 polypeptide ormodified p35 polypeptide includes any oligodeoxynucleotide oroligodeoxyribonucleotide sequence which, upon expression in anappropriate host cell, results in production of a modified p40polypeptide or modified p35 polypeptide of the invention. The DNA or RNAcan be produced in an appropriate host cell, or in a cell-free (invitro) system, or can be produced synthetically (e.g., by anamplification technique such as PCR) or chemically.

Using Polynucleotides of the Invention

The polynucleotides of the invention have a variety of uses in, forexample: recombinant production (i.e., expression) of the modified p40polypeptides and modified p35 polypeptides of the invention; astherapeutics and prophylactics, e.g., for use in methods of therapeuticand prophylactic treatment of a disease, disorder or condition; for usein, gene therapy methods and related applications; as immunogens; foruse in diagnostic and screening assays; as diagnostic probes for thepresence of complementary or partially complementary nucleic acids(including for detection of nucleic acids encoding modified p40polypeptides or modified p35 polypeptides); as substrates for furtherreactions, e.g., recursive recombination or mutation reactions toproduce new and/or improved modified p40 polypeptides or modified p35polypeptides, and the like.

Expression of Polypeptides of the Invention

In accordance with the present invention, polynucleotide sequences whichencode modified p35 polypeptides or modified p40 polypeptides (in matureform or comprising a leader peptide sequence), fragments of thepolypeptides, related fusion proteins, or functional equivalentsthereof, are collectively referred to herein as “modified cytokinepolypeptides”, “modified p35 polypeptides”, “modified p40 polypeptides”or, simply, “polypeptides of the invention”. Polypeptide or amino acidfragments of each of the preceding terms are also intended to beincluded and encompassed in the polypeptides or proteins of theinvention. Such polynucleotide sequences of the invention are used inrecombinant DNA (or RNA) molecules that direct the expression of themodified p40 polypeptides and the modified p35 polypeptides inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other nucleic acid sequences which encode substantially the sameor a functionally equivalent amino acid sequence are also used to cloneand express the modified p40 polypeptides and the modified p35polypeptides.

Modified Coding Sequences

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence (including, e.g., a nucleotidesequence encoding a modified p40 or modified p35 polypeptide of theinvention or a fragment thereof) to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms preferentially use a subset of these codons. Thecodons that are utilized most often in a species are called optimalcodons, and those not utilized very often are classified as rare orlow-usage codons (see, e.g., Zhang S P et al. (1991) Gene 105:61-72).Codons can be substituted to reflect the preferred codon usage of thehost, a process called “codon optimization” or “controlling for speciescodon bias.”

Optimized coding sequence containing codons preferred by a particularprokaryotic or eukaryotic host (see also Murray, E. et al. (1989) Nuc.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,preferred stop codons for S. cerevisiae and mammals are UAA and UGArespectively. The preferred stop codon for monocotyledonous plants isUGA, whereas insects and E. coli prefer to use UAA as the stop codon(Dalphin M E et al. (1996) Nuc. Acids Res. 24: 216-218).

The polynucleotide sequences of the present invention can be engineeredin order to alter a modified p35 or modified p40 coding sequence for avariety of reasons, including but not limited to, alterations whichmodify the cloning, processing and/or expression of the gene product.For example, alterations may be introduced using techniques which arewell known in the art, e.g., site-directed mutagenesis, to insert newrestriction sites, to alter glycosylation patterns, to change codonpreference, to introduce splice sites, etc.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the nucleic acid sequences as broadly described herein(e.g., those encoding a modified p40 or modified p35 polypeptide of theinvention or a fragment thereof). The constructs comprise a vector, suchas, a plasmid, a cosmid, a phage, a virus, a bacterial artificialchromosome (BAC), a yeast artificial chromosome (YAC), and the like,into which a nucleic acid sequence of the invention has been inserted,in a forward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

General texts which describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Juo, P-S., CONCISE DICTIONARY OF BIOMEDICAL ANDMOLECULAR BIOLOGY (CRC Press 1996); Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGEDICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); Hale & Marham,THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991); Scott and Mercer,CONCISE ENCYCLOPEDIA OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (3d ed.1997); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology, volume 152 Academic Press, Inc., San Diego, Calif.(hereinafter “Berger”); Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989 (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.(supplemented through 1999) (“Ausubel”)). Examples of techniquessufficient to direct persons of skill through in vitro amplificationmethods, including the polymerase chain reaction (PCR), the ligase chainreaction (LCR), Qβ-replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA), e.g., for the production of thehomologous nucleic acids of the invention are found in Berger, Sambrook,and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202;U.S. Pat. No. 4,683,195, issued Jul. 28, 1997; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Nat'l Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Nat'lAcad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem. 35, 1826;Landegren et al. (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13:563-564.

PCR generally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA, and/or DNA, are amplified by methods wellknown in the art (see, e.g., U.S. Pat. No. 4,683,195 and otherreferences above). Generally, sequence information from the ends of theregion of interest or beyond is used, for design of oligonucleotideprimers. Such primers will be identical or similar in sequence to theopposite strands of the template to be amplified. The 5′ terminalnucleotides of the opposite strands may coincide with the ends of theamplified material. PCR may be used to amplify specific RNA or specificDNA sequences, recombinant DNA or RNA sequences, DNA and RNA sequencesfrom total genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. PCR is one example, but not theonly example, of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a another(e.g., known) nucleic acid as a primer. Improved methods of cloning invitro amplified nucleic acids are described in Wallace et al., U.S. Pat.No. 5,426,039. Improved methods of amplifying large nucleic acids by PCRare summarized in Cheng et al. (1994) Nature 369:684-685 and thereferences therein, in which PCR amplicons of up to 40 kb are generated.One of skill will appreciate that essentially any RNA can be convertedinto a double stranded DNA suitable for restriction digestion, PCRexpansion and sequencing using reverse transcriptase and a polymerase.See Ausubel, Sambrook and Berger, all supra.

The present invention also relates to host cells which are transducedwith vectors of the invention, and the production of polypeptides of theinvention (including fragments thereof) by recombinant techniques. Hostcells are genetically engineered (i.e., transduced, transformed ortransfected) with the vectors of this invention, which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the modified p40 or modified p35 gene. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art and in the references cited herein,including, e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, 3d ed., Wiley-Liss, New York and the references citedtherein.

The modified p40 and modified p35 polypeptides and proteins of theinvention can also be produced in non-animal cells such as plants,yeast, fungi, bacteria and the like. In addition to Sambrook, Berger andAusubel, details regarding cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods, Springer LabManual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks(eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla.

The polynucleotides of the present invention may be included in any oneof a variety of expression vectors for expressing a polypeptide. Suchvectors include chromosomal, nonchromosomal and synthetic DNA sequences,e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus;yeast plasmids; vectors derived from combinations of plasmids and phageDNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,pseudorabies, adenovirus, adeno-associated virus, retroviruses and manyothers. Any vector that transducers genetic material into a cell, and ifreplication is desired, which is replicable and viable in the relevanthost can be used.

The nucleic acid sequence in the expression vector is operatively linkedto an appropriate transcription control sequence (promoter) to directmRNA synthesis. Examples of such promoters include: LTR or SV40promoter, E. coli lac or trp promoter, phage lambda P_(L) promoter, andother promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector also contains aribosome binding site for translation initiation, and a transcriptionterminator. The vector optionally includes appropriate sequences foramplifying expression. In addition, the expression vectors optionallycomprise one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells, such as dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as described herein,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express theprotein. Examples of appropriate expression hosts include: bacterialcells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungalcells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurosporacrassa; insect cells such as Drosophila and Spodoptera frugiperda;mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma; plantcells, etc. It is understood that not all cells or cell lines need to becapable of producing fully functional modified p40 or modified p35polypeptides; for example, antigenic fragments of a modified p40 ormodified p35 polypeptide may be produced in a bacterial or otherexpression system. The invention is not limited by the host cellsemployed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the polypeptide of the invention.For example, when large quantities of modified p40 or modified p35polypeptide or fragments thereof are needed for the induction ofantibodies, vectors which direct high level expression of fusionproteins that are readily purified may be desirable. Such vectorsinclude, but are not limited to, multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which themodified p40 or modified p35 coding sequence may be ligated into thevector in-frame with sequences for the amino-terminal Met and thesubsequent 7 residues of beta-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem.264:5503-5509); pET vectors (Novagen, Madison Wis.); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of the modified p40and/or modified p35 polypeptides of the invention. For reviews, seeAusubel et al. (supra) and Grant et al. (1987; Methods in Enzymology153:516-544).

In mammalian host cells, a number expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome will result in a viablevirus capable of expressing modified p40 or modified p35 polypeptide ininfected host cells (Logan and Shenk (1984) Proc. Natl. Acad. Sci.81:3655-3659). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Additional Expression Elements

Specific initiation signals can aid in efficient translation of amodified p35 or modified p40 coding sequence. These signals can include,e.g., the ATG initiation codon and adjacent sequences. In cases where acoding sequence, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence (e.g., a mature protein coding sequence), or aportion thereof, is inserted, exogenous transcriptional control signalsincluding the ATG initiation codon must be provided. Furthermore, theinitiation codon must be in the correct reading frame to ensuretranscription of the entire insert. Exogenous transcriptional elementsand initiation codons can be of various origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof enhancers appropriate to the cell system in use (Scharf D et al.(1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987)Methods in Enzymol 153:516-544).

Secretion/Localization Sequences

Polynucleotides of the invention can also be fused, for example,in-frame to nucleic acid encoding a secretion/localization sequence, totarget polypeptide expression to a desired cellular compartment,membrane, or organelle, or to direct polypeptide secretion to theperiplasmic space or into the cell culture media. Such sequences areknown to those of skill, and include secretion leader peptides,organelle targeting sequences (e.g., nuclear localization sequences, ERretention signals, mitochondrial transit sequences, chloroplast transitsequences), membrane localization/anchor sequences (e.g., stop transfersequences, GPI anchor sequences), and the like. Polypeptides expressedby such polynucleotides of the invention may include the amino acidsequence corresponding to the secretion and/or localization sequence(s).

Expression Hosts

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be aeukaryotic cell, such as a mammalian cell, a yeast cell, or a plantcell, or the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,electroporation, or other common techniques (Davis, L., Dibner, M., andBattey, I. (1986) Basic Methods in Molecular Biology). The cell mayinclude a nucleic acid of the invention, said nucleic acid encoding apolypeptide, wherein said cells expresses a polypeptide (e.g., amodified p40 polypeptide or modified p35 polypeptide having T-cellproliferative activity, or interferon-gamma induction activity inT-cells, as measured by the assays described herein). The invention alsoincludes a vector comprising any nucleic acid of the invention describedherein and includes a cell transduced by such a vector. Furthermore,cells and transgenic animals which include any polypeptide or nucleicacid above or throughout this specification, e.g., produced bytransduction of a vector of the invention, are an additional feature ofthe invention.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingwhich cleaves a “pre” or a “prepro” form of the protein may also beimportant for correct insertion, folding and/or function. Different hostcells such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression can be used. For example, cell lines which stably express apolypeptide of the invention are transduced using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced sequences. For example, resistant clumps of stablytransformed cells can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleic acid sequence encoding apolypeptide of the invention are optionally cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. The protein or fragment thereof produced by a recombinantcell may be secreted, membrane-bound, or contained intracellularly,depending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containingpolynucleotides encoding mature proteins of the invention can bedesigned with leader peptide (also termed “signal peptide”) sequenceswhich direct secretion of the mature polypeptides through a prokaryoticor eukaryotic cell membrane.

Additional Polypeptide Sequences

The polynucleotides of the present invention may also comprise a codingsequence fused in-frame to a marker sequence which, e.g., facilitatespurification of the encoded polypeptide. Such purification facilitatingdomains include, but are not limited to, metal chelating peptides suchas histidine-tryptophan modules that allow purification on immobilizedmetals, a sequence which binds glutathione (e.g., GST), a hemagglutinin(HA) tag (corresponding to an epitope derived from the influenzahemagglutinin protein; Wilson, I., et al. (1984) Cell 37:767), maltosebinding protein sequences, a FLAG epitope utilized in the FLAGSexpression/affinity purification system (Immunex Corp, Seattle, Wash.),an E-epitope tag (E-tag), and the like. The inclusion of aprotease-cleavable polypeptide linker sequence between the purificationdomain and the polypeptide sequence is useful to facilitatepurification. One expression vector contemplated for use in thecompositions and methods described herein provides for expression of afusion protein comprising a polypeptide of the invention fused to apolyhistidine region separated by an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography, as described in Porath et al. (1992)Protein Expression and Purification 3:263-281) while the enterokinasecleavage site provides a means for separating the polypeptide from thefusion protein. pGEX vectors (Promega; Madison, Wis.) may also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption to ligand-agarosebeads (e.g., glutathione-agarose in the case of GST-fusions) followed byelution in the presence of free ligand.

Polypeptide Production and Recovery

Following transduction of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well know to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, Third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) MAMMALIAN CELLCULTURE: ESSENTIAL TECHNIQUES John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Plant Molecular Biology (1993) R. R. D. Croy,Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cellculture media in general are set forth in Atlas and Parks (eds) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue(1998) from Sigma-Aldrich, Inc. (St. Louis, Mo.) (“Sigma-LSRCCC”) and,e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St. Louis, Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted supra, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al.(1996) Protein Methods, 2^(nd) Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ; Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

In Vitro Expression Systems

Cell-free transcription/translation systems can also be employed toproduce polypeptides using DNAs or RNAs of the present invention.Several such systems are commercially available. A general guide to invitro transcription and translation protocols is found in Tymms (11995)In vitro Transcription and Translation Protocols: Methods in MolecularBiology (Volume 37), Garland Publishing, NY.

Modified Amino Acids: Polypeptides of the invention may contain one ormore modified amino acid. The presence of modified amino acids may beadvantageous in, for example, (a) increasing polypeptide serumhalf-life, (b) reducing polypeptide antigenicity, (c) increasingpolypeptide storage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means.

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEG-ylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, N.J.

In Vitro, In Vivo, and Ex Vivo Uses of Polynucleotides and Polypeptidesof the Invention

The polynucleotides and polypeptides of the invention have a variety ofuses, including, but not limited to, for example: in recombinantproduction (i.e., expression) of the recombinant modified p40polypeptides and modified p35 polypeptides of the invention; astherapeutic and prophylactic agents in methods of in vivo and ex vivotreatment of a variety of diseases, disorders, and conditions in avariety of subjects; for use in in vitro methods, such as diagnostic andscreening methods, to detect, diagnose, and treat a variety of diseases,disorders, and conditions (e.g., cancers, viral-based disorders,angiogenic-based disorders) in a variety of subjects (e.g., mammals); asimmunogens; in gene therapy methods and DNA- or RNA-based deliverymethods to deliver or administer in vivo, ex vivo, or in vitrobiologically active polypeptides of the invention to a tissue,population or cells, organ, graft, bodily system of a subject (e.g.,organ system, lymphatic system, blood system, etc.); as DNA vaccines,multi-component vaccines for use in prophylactic or therapeutictreatment of a variety of diseases, disorders, or other conditions(e.g., cancers, viral-based disorders, angiogenic-based disorders) in avariety of subjects (e.g., mammals); as adjuvants to enhance or augmentan immune response in a subject; as a component of a multiple-stepboosting vaccination method (e.g., a format comprising a primevaccination by delivery of a DNA or RNA nucleotide (e.g., a nucleotideencoding a polypeptide of the invention or encoding another polypeptide)followed by a second boost of a polypeptide (e.g., a polypeptide of theinvention or other polypeptide); as diagnostic probes for the presenceof complementary or partially complementary nucleic acids (including fordetection of naturally-occurring p40 and p35 coding nucleic acids); assubstrates for further reactions, e.g., shuffling reactions, mutationreactions, or other diversity generation reactions to produce new and/orimproved modified p40 polypeptides and modified p35 polypeptides and newmodified p40 nucleic acids and modified p35 nucleic acids encoding suchpolypeptides, e.g., to evolve novel therapeutic or prophylacticproperties, and the like; for polymerase chain reactions (PCR) orcloning methods, e.g., including digestion or ligation reactions, toidentify new and/or improved naturally-occurring or non-naturallyoccurring p40 or p35 nucleic acids and polypeptides encoded therefrom.Polynucleotides which encode a modified p40 polypeptide or modified p35polypeptide, or complements of the polynucleotides, are optionallyadministered to a cell to accomplish a therapeutically orprophylactically useful process or to express a therapeutically usefulproduct in vivo, ex vivo, or in vitro. These applications, including invivo or ex vivo applications, including, e.g., gene therapy, include amultitude of techniques by which gene expression may be altered incells. Such methods include, for instance, the introduction of genes forexpression of, e.g., therapeutically or prophylactically usefulpolypeptides, such as the modified p40 polypeptides and modified p35polypeptides of the present invention. Such methods include, forexample, infecting with a retrovirus comprising the polynucleotidesand/or polypeptides of the invention. Optionally, the retrovirus furthercomprises additional exogenous, e.g., therapeutic or prophylactic geneconstruct, sequences. In one aspect, the invention provides gene therapymethods of prophylactically or therapeutically treating a disease,disorder or condition in a subject in need of such treatment byadministering in vivo, ex vivo, or in vitro one or more nucleic acids ofthe invention described herein to one or more cells of a subject,including an organism or mammal, including, e.g., a human, primate,mouse, dog, cat, pig, cow, goat, rabbit, rat, guinea pig, hamster,horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., achicken or duck) or a fish, or invertebrate, as described in more detailbelow.

In another aspect, the invention provides methods of prophylactically ortherapeutically treating a disease, disorder or condition in a subjectin need of such treatment by administering in vivo, ex vivo, or in vitroone or more polypeptides of the invention described herein to one ormore cells of a subject (including those defined herein), as describedin more detail below.

Polypeptide Expression

Polynucleotides encoding polypeptides of the invention are particularlyuseful for in vivo or ex vivo therapeutic or prophylactic applications,using techniques well known to those skilled in the art. For example,cultured cells are engineered ex vivo with a polynucleotide (DNA orRNA), with the engineered cells then being returned to the patient.Cells may also be engineered in vivo or ex vivo for expression of apolypeptide in vivo or ex vivo, respectively.

A number of viral vectors suitable for organismal in vivo transductionand expression are known. Such vectors include retroviral vectors (seeMiller, Curr. Top. Microbiol. Immunol. (1992) 158:1-24; Salmons andGunzburg, (1993) Human Gene Therapy 4:129-141; Miller et al., (1994)Methods in Enzymology 217: 581-599) and adeno-associated vectors(reviewed in Carter (1992) Curr. Opinion Biotech. 3: 533-539; Muzcyzka(1992) Curr. Top. Microbiol. Immunol. 158: 97-129). Other viral vectorsthat are used include adenoviral vectors, herpes viral vectors andSindbis viral vectors, as generally described in, e.g., Jolly (1994)Cancer Gene Therapy 1:51-64; Latchman (1994) Molec. Biotechnol.2:179-195; and Johanning et al., (1995) Nucl. Acids Res. 23:1495-1501.

Gene therapy provides methods for combating chronic infectious diseases(e.g., HIV infection, viral hepatitis), as well as non-infectiousdiseases including cancer and allergic diseases and some forms ofcongenital defects such as enzyme deficiencies. Several approaches forintroducing nucleic acids into cells in vivo, ex vivo and in vitro havebeen used. These include liposome based gene delivery (Debs and Zhu(1993) WO 93/24640 and U.S. Pat. No. 5,641,662; Mannino andGould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose, U.S. Pat. No.5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc.Natl. Acad. Sci. USA 84: 7413-7414); Brigham, et al. (1989) Am. J. Med.Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288;Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; andWang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855);adenoviral vector mediated gene delivery, e.g., to treat cancer (see,e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057; Tonget al. (1996). Gynecol. Oncol. 61: 175-179; Clayman et al. (1995) CancerRes. 5: 1-6; O'Malley et al. (1995) Cancer Res. 55: 1080-1085; Hwang etal. (1995) Am. J. Respir. Cell Mol. Biol. 13: 7-16; Haddada et al.(1995) Curr. Top. Microbiol. Immunol. 199 (Pt. 3): 297-306; Addison etal. (1995) Proc. Nat'l. Acad. Sci. USA 92: 8522-8526; Colak et al.(1995) Brain Res. 691: 76-82; Crystal (1995) Science 270: 404-410;Elshami et al. (1996) Human Gene Ther. 7: 141-148; Vincent et al. (1996)J. Neurosurg. 85: 648-654), and many other diseases.Replication-defective retroviral vectors harboring therapeuticpolynucleotide sequence as part of the retroviral genome have also beenused, particularly with regard to simple MuLV vectors. See, e.g., Milleret al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J. NIHRes. 4:43, and Cornetta et al. (1991) Hum. Gene Ther. 2:215). Nucleicacid transport coupled to ligand-specific, cation-based transportsystems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) have alsobeen used Naked DNA expression vectors have also been described (Nabelet al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Ingeneral, these approaches can be adapted to the invention byincorporating nucleic acids encoding the polypeptides of the inventioninto the appropriate vectors.

General texts which describe gene therapy protocols, which can beadapted to the present invention by introducing the nucleic acids of theinvention into patients, include Robbins (1996) Gene Therapy Protocols,Humana Press, NJ, and Joyner (1993) Gene Targeting: A PracticalApproach, IRL Press, Oxford, England.

Antisense Technology

In addition to expression of the nucleic acids of the invention as genereplacement nucleic acids, the nucleic acids are also useful for senseand anti-sense suppression of expression, e.g., to down-regulateexpression of a nucleic acid of the invention, once expression of thenucleic acid is no-longer desired in the cell. Similarly, the nucleicacids of the invention, or subsequences or anti-sense sequences thereof,can also be used to block expression of naturally occurring homologousnucleic acids. A variety of sense and anti-sense technologies are knownin the art, e.g., as set forth in Lichtenstein and Nellen (1997)Antisense Technology: A Practical Approach IRL Press at OxfordUniversity, Oxford, England, and in Agrawal (1996) AntisenseTherepeutics Humana Press, NJ, and the references cited therein.

Pharmaceutical Compositions

The polynucleotides of the invention (including vectors, cells,antibodies, etc., comprising polynucleotides or polypeptides of theinvention) may be employed for therapeutic uses in combination with asuitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. A pharmaceutically acceptable carrierencompasses any of the standard pharmaceutical carriers, buffers andexcipients. Such a carrier or excipient includes, but is not limited to,saline, buffered saline (e.g., phosphate-buffered saline solution),dextrose, water, glycerol, ethanol, emulsions (such as an oil/water orwater/oil emulsion), various types of wetting agents and/or adjuvants,and combinations thereof. Suitable pharmaceutical carriers and agentsare described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack PublishingCo., Easton, 19^(th) ed. 1995). The formulation should suit the mode ofadministration of the active agent (e.g., nucleotide, polypeptide,vector, cell, etc.). Methods of administering nucleic acids,polypeptides, vectors, cells, antibodies, and proteins are well known inthe art, and further discussed below.

Use as Probes

Also contemplated are uses of polynucleotides, also referred to hereinas oligonucleotides, typically having at least 12 bases, preferably atleast 15, more preferably at least 20, 30, or 50 bases, which hybridizeunder at least highly stringent (or ultra-high stringent orultra-ultra-high stringent conditions) to a modified p40 or modified p35polynucleotide sequence of the invention described herein. Thepolynucleotides may be used as probes, primers, sense and antisenseagents, and the like, according to methods as noted supra.

Sequence Variations

Silent Variations

It will be appreciated by those skilled in the art that due to thedegeneracy of the genetic code, a multitude of nucleic acids sequencesencoding polypeptides of the invention may be produced, some which maybear minimal sequence homology to the nucleic acid sequences explicitlydisclosed herein. TABLE 4 Codon Table Amino acids Codon Alanine Ala AGCA GCC GCG GCU Cysteine Gys C UGC UGU Aspartic acid Asp D GAC GAUGlutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly GGGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUULysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU MethionineMet M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCUGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SerineSer S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine ValV GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For instance, inspection of the codon table (Table 4) shows that codonsAGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.Thus, at every position in the nucleic acids of the invention where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described above without altering the encodedpolypeptide. It is understood that U in an RNA sequence corresponds to Tin a DNA sequence.

Using, as an example, the DNA sequence corresponding to nucleotides 1-15of SEQ ID NO:1, ATGTGTCACCAGCAG, an example of a silent variation ofthis sequence is ATGTGCCATCAACAA (SEQ ID NO:41), both sequences whichencode the amino acid sequence MCHQQ, corresponding to amino acids 1-5of SEQ ID NO:8.

Such “silent variations” are one species of “conservatively modifiedvariations”, discussed below. One of skill will recognize that eachcodon in a nucleic acid (except AUG and UGC, which are ordinarily theonly codons for methionine and tryptophan, respectively) can be modifiedby standard techniques to encode a functionally identical polypeptide.Accordingly, each silent variation of a nucleic acid which encodes apolypeptide is implicit in any described sequence. The inventionprovides each and every possible variation of nucleic acid sequenceencoding a polypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table 4) as applied to the nucleic acid sequence encoding apolypeptide of the invention. All such variations of every nucleic acidherein are specifically provided and described by consideration of thesequence in combination with the genetic code.

Conservative Variations

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than 5%, more typically less than 4%, 2% or 1%) inan encoded sequence are “conservatively modified variations” where thealterations result in a deletion of an amino acid, an addition of anamino acid, or a substitution of an amino acid with a chemically similaramino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table 5 sets forth six groups whichcontain amino acids that are “conservative substitutions” for oneanother. TABLE 5 Conservative Amino Acid Substitution Groups Group 1Alanine Serine Threonine (A) (S) (T) Group 2 Aspartic acid Glutamic acid(D) (E) Group 3 Asparagine Glutamine (N) (Q) Group 4 Arginine Lysine (R)(K) Group 5 Isoleucine Leucine Methionine Valine (I) (L) (M) (V) Group 6Phenylalanine Tyrosine Tryptophan (F) (Y) (W)

Thus, “conservatively substituted variations” of a listed polypeptidesequence of the present invention include substitutions of a smallpercentage, typically less than 5%, more typically less than 2% or 1%,of the amino acids of the polypeptide sequence, with a conservativelyselected amino acid of the same conservative substitution group.

For example, a conservatively substituted variation of the polypeptideidentified herein as SEQ ID NO:8 will contain “conservativesubstitutions”, according to the six groups defined above, in up toabout 16 residues (i.e., about 5% of the amino acids) in the 325 aminoacid polypeptide.

In a further example, examples of conservatively substituted variationsof the region corresponding to amino acids 23-47 of SEQ ID NO:8,

IWELK KDVYV VELDW YPNAP GETVV include:

IWDLK RDVYV IELDW FPNAP GETLV (SEQ ID NO:42) and

VWEIK KDMYV VELEW YPNAP GETVI 0 (SEQ ID NO:43), and the like,

in accordance with the exemplary conservative substitutions listed inTable 5 (in the above example, conservative substitutions areunderlined). Listing of a polypeptide sequence herein, in conjunctionwith the above substitution table, provides an express listing of allconservatively substituted polypeptides.

Finally, the addition of sequences which do not alter the encodedactivity of a nucleic acid molecule, such as the addition of anon-functional sequence, is a conservative variation of the basicnucleic acid.

One of ordinary skill will appreciate that many conservative variationsof the nucleic acid constructs which are disclosed yield a functionallyidentical construct. For example, as discussed above, owing to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

Nucleic Acid Hybridization

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Ausubel, supra, Hames and Higgins (1995) Gene Probes 1, IRL Press atOxford University Press, Oxford, England (Hames and Higgins 1) and Hamesand Higgins (1995) Gene Probes 2, IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments, such as Southern and northern hybridizations,are sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra, and in Hames and Higgins 1 and Hames andHiggins 2, supra.

For purposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. or lesslower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH (as noted below, highly stringentconditions can also be referred to in comparative terms). The T_(m) isthe temperature (under defined ionic strength and pH) at which 50% ofthe test sequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the T_(m) for aparticular probe.

The T_(m) is the temperature of the nucleic acid duplexes indicates thetemperature at which the duplex is 50% denatured under the givenconditions and its represents a direct measure of the stability of thenucleic acid hybrid. Thus, the T_(m) corresponds to the temperaturecorresponding to the midpoint in transition from helix to random coil;it depends on length, nucleotide composition, and ionic strength forlong stretches of nucleotides.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the hybridization temperature) lowers the backgroundsignal, typically with only the specific signal remaining. See Rapley,R. and Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,Inc. 1998) (hereinafter “Rapley and Walker”), which is incorporatedherein by reference in its entirety for all purposes.

The T_(m) of a DNA-DNA duplex can be estimated using Equation 1 asfollows:T _(m)(° C.)=81.5° C.+16.6 (log₁₀M)+0.41 (% G+C)−0.72 (% f)−500/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formalize and n is the number of nucleotide bases(i.e., length) of the hybrid. See Rapley and Walker, supra.

The T_(m) of an RNA-DNA duplex can be estimated by using Equation 2 asfollows:T _(m)(° C.)=79.8° C.+18.5 (log₁₀M)+0.58 (% G+C)−11.8(% G+C)²−0.56 (%f)−820/n,where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Id.

Equations 1 and 2 are typically accurate only for hybrid duplexes longerthan about 100-200 nucleotides. Id.

The T_(m) of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:T _(m)(° C.)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes.

In general, a signal to noise ratio of 2.5×-5× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization. Detection of at leaststringent hybridization between two sequences in the context of thepresent invention indicates relatively strong structural similarity orhomology to, e.g., the nucleic acids of the present invention providedin the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringency conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary. See, e.g., Rapley and Walker, supra.

Comparative hybridization can be used to identify nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. Detection ofhighly stringent hybridization between two nucleotide sequences in thecontext of the present invention indicates relatively strong structuralsimilarity/homology to, e.g., the nucleic acids provided in the sequencelisting herein. Highly stringent hybridization between two nucleotidesequences demonstrates a degree of similarity or homology of structure,nucleotide base composition, arrangement or order that is greater thanthat detected by stringent hybridization conditions. In particular,detection of highly stringent hybridization in the context of thepresent invention indicates strong structural similarity or structuralhomology (e.g., nucleotide structure, base composition, arrangement ororder) to, e.g., the nucleic acids provided in the sequence listingsherein. For example, it is desirable to identify test nucleic acidswhich hybridize to the exemplar nucleic acids herein under stringentconditions.

Thus, one measure of stringent hybridization is the ability to hybridizeto one of the listed nucleic acids (e.g., nucleic acid sequences SEQ IDNO:1 to SEQ ID NO:7 and SEQ ID NO:16 to SEQ ID NO:25, and complementarypolynucleotide sequences thereof), under highly stringent conditions (orvery stringent conditions, or ultra-high stringency hybridizationconditions, or ultra-ultra high stringency hybridization conditions).Stringent hybridization (as well as highly stringent, ultra-highstringency, or ultra-ultra high stringency hybridization conditions) andwash conditions can easily be determined empirically for any testnucleic acid. For example, in determining highly stringent hybridizationand wash conditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom SEQ ID NO:1 to SEQ ID NO:7 and SEQ ID NO:16 to SEQ ID NO:25, andcomplementary polynucleotide sequences thereof, binds to a perfectlymatched complementary target (again, a nucleic acid comprising one ormore nucleic acid sequences selected from SEQ ID NO:1 to SEQ ID NO:7 andSEQ ID NO:16 to SEQ ID NO:25, and complementary polynucleotide sequencesthereof), with a signal to noise ratio that is at least about 2.5×, andoptionally about 5× or more as high as that observed for hybridizationof the probe to an unmatched target. In this case, the unmatched targetis a nucleic acid corresponding to a known p40 or p35, e.g., a p40 orp35 nucleic acid that is present in a public database such as GenBank atthe time of filing of the subject application. Examples of suchunmatched target nucleic acids include, e.g., p40 nucleic acids with thefollowing GenBank accession numbers: M65272 and M65290 (human), U19841(Macaca mulatta, rhesus monkey), U19834 (Cercocebus torquatus, sootymangabey), Y11129 (Equus caballus, horse), U83184, Y07762 and AF054607(Felis catus, cat), U49100 and AF091134 (Canis familiaris, dog), U57752and U10160 (Cervus elaphus, red deer), AF007576 (Capra hircus, goat),AF004024 (Ovis aries, sheep), U11815 (Bos taurus, cow), U08317 (Susscrofa, pig), X97019 and AF082494 (Marmota monax, woodchuck), AF133197and U16674 (Rattus norvegicus, rat), M86671 and S82426 (Mus musculus,mouse), AF097507 (Cavia porcellus, guinea pig), and AF046211(Mesocricetus auratus, golden hamster). Additional such sequences can beidentified in e.g., GenBank, by one of ordinary skill in the art.

Examples of such unmatched target nucleic acids also include, e.g., p35nucleic acids having the following GenBank accession numbers: M65271,M65291 (human); U19842 (Macaca mulatta, rhesus monkey), U19835(Cercocebus torquatus, sooty mangabey), U83185, Y07761, AF054605 (Feliscatus, cat), U49085 (Canis familiaris, dog), L35765 (Sus scrofa, pig),Y11130 (Equus caballus, horse), U14416 (Bos taurus, cow), U57751 (Cervuselaphus, red deer), AF173557 (Ovis aries, sheep), AF003542 (Caprahircus, goat), X97018 (Marmota monax, woodchuck), AF177031 (Rattusnorvegicus, rat), and M86672, S82419 (Mus musculus, mouse). Additionalsuch sequences can be identified in public databases, e.g., GenBank, byone of ordinary skill in the art.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 2.5×-10×, typically about 5×-10× as high as thatobserved for hybridization to any of the unmatched p40 target nucleicacids represented by GenBank accession numbers M65272 and M65290(human), U19841 (Macaca mulatta, rhesus monkey), U19834 (Cercocebustorquatus, sooty mangabey), Y11129 (Equus caballus, horse), U83184,Y07762 and AF054607 (Felis catus, cat), U49100 and AF091134 (Canisfamiliaris, dog), U57752 and U10160 (Cervus elaphus, red deer), AF007576(Capra hircus, goat), AF004024 (Ovis aries, sheep), U11815 (Bos taurus,cow), U08317 (Sus scrofa, pig), X97019 and AF082494 (Marmota monax,woodchuck), AF133197 and U16674 (Rattus norvegicus, rat), M86671 andS82426 (Mus musculus, mouse), AF097507 (Cavia porcellus, guinea pig),and AF046211 (Mesocricetus auratus, golden hamster), or to any of theunmatched p35 target nucleic acids represented by GenBank accessionnumbers M65271, M65291 (human); U19842 (Macaca mulatta, rhesus monkey),U19835 (Cercocebus torquatus, sooty mangabey), U83185, Y07761, AF054605(Felis catus, cat), U49085 (Canis familiaris, dog), L35765 (Sus scrofa,pig), Y11130 (Equus caballus, horse), U14416 (Bos taurus, cow), U57751(Cervus elaphus, red deer), AF173557 (Ovis aries, sheep), AF003542(Capra hircus, goat), X97018 (Marmota monax, woodchuck), AF177031(Rattus norvegicus, rat), and M86672, S82419 (Mus musculus, mouse).

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched p40 orp35 target nucleic acids described above. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least ½ that of the perfectly matched complementary targetnucleic acid is said to bind to the probe under ultra-high stringencyconditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched p40 or p35 target nucleic acids described above can beidentified. A target nucleic acid which hybridizes to a probe under suchconditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-ultra-high stringency conditions.

Target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NO:1 to SEQ ID NO:7, and SEQ ID NO:16 to SEQ ID NO:25 under high,ultra-high and ultra-ultra high stringency conditions are a feature ofthe invention. Examples of such nucleic acids include those with one ora few silent or conservative nucleic acid substitutions as compared to agiven nucleic acid sequence.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code, or when antisera or antiserum generatedagainst one or more of SEQ ID NO:8 to SEQ ID NO:14, which has beensubtracted using the polypeptides encoded by known p40 sequences,including, e.g., the those encoded by the following p40 nucleic acidsequences in GenBank: Accession numbers: M65272 and M65290 (human),U19841 (Macaca mulatta, rhesus monkey), U19834 (Cercocebus torquatus,sooty mangabey), Y11129 (Equus caballus, horse), U83184, Y07762 andAF054607 (Felis catus, cat), U49100 and AF091134 (Canis familiaris,dog), U57752 and U10160 (Cervus elaphus, red deer), AF007576 (Caprahircus, goat); AF004024 (Ovis aries, sheep), U11815 (Bos taurus, cow),U08317 (Sus scrofa, pig), X97019 and AF082494 (Marmota monax,woodchuck), AF133197 and U16674 (Rattus norvegicus, rat), M86671 andS82426 (Mus musculus, mouse), AF097507 (Cavia porcellus, guinea pig),and AF046211 (Mesocricetus auratus, golden hamster), or other similarp40 sequences presented in GenBank, or when antisera generated againstone or more of SEQ ID NO:26 to SEQ ID NO:35, which has been subtractedusing the polypeptides encoded by known p35 sequences, including, e.g.,the those encoded by the following p35 nucleic acid sequences inGenBank: Accession numbers: M65271, M65291 (human); U19842 (Macacamulatta, rhesus monkey), U19835 (Cercocebus torquatus, sooty mangabey),U83185, Y07761, AF054605 (Felis catus, cat), U49085 (Canis familiaris,dog), L35765 (Sus scrofa, pig), Y11130 (Equus caballus, horse), U14416(Bos taurus, cow), U57751 (Cervus elaphus, red deer), AF173557 (Ovisaries, sheep), AF003542 (Capra hircus, goat), X97018 (Marmota monax,woodchuck), AF177031 (Rattus norvegicus, rat), and M86672, S82419 (Musmusculus, mouse), or other similar p35 sequences presented in GenBank.Further details on immunological identification of polypeptides of theinvention are found below. Additionally, for distinguishing betweenduplexes with sequences of less than about 100 nucleotides, a TMAC1hybridization procedure known to those of ordinary skill in the art canbe used. See, e.g., Sorg, U. et al. 1 Nucleic Acids Res. (Sep. 11, 1991)19(17), incorporated herein by reference in its entirety for allpurposes.

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from SEQ ID NO:1 to SEQ IDNO:7, or SEQ ID NO: 16 to SEQ ID NO:25. The unique subsequence is uniqueas compared to a nucleic acid corresponding to any known p40 nucleicacid sequence including, e.g., the known sequences represented byGenBank accession numbers: M65272 and M65290 (human), U19841 (Macacamulatta, rhesus monkey), U19834 (Cercocebus torquatus, sooty mangabey),Y11129 (Equus caballus, horse), U83184, Y07762 and AF054607 (Feliscatus, cat), U49100 and AF091134 (Canis familiaris, dog), U57752 andU10160 (Cervus elaphus, red deer), AF007576 (Capra hircus, goat),AF004024 (Ovis aries, sheep), U11815 (Bos taurus, cow), U08317 (Susscrofa, pig), X97019 and AF082494 (Marmota monax, woodchuck), AF133197and U16674 (Rattus norvegicus, rat), M86671 and S82426 (Mus musculus,mouse), AF097507 (Cavia porcellus, guinea pig), and AF046211(Mesocricetus auratus, golden hamster); or unique as compared to anucleic acid corresponding to any known p35 nucleic acid sequenceincluding, e.g., the known sequences represented by GenBank accessionnumbers M65271, M65291 (human); U19842 (Macaca mulatta, rhesus monkey),U19835 (Cercocebus torquatus, sooty mangabey), U83185, Y07761, AF054605(Felis catus, cat), U49085 (Canis familiaris, dog), L35765 (Sus scrofa,pig), Y11130 (Equus caballus, horse), U14416 (Bos taurus, cow), U57751(Cervus elaphus, red deer), AF173557 (Ovis aries, sheep), AF003542(Capra hircus, goat), X97018 (Marmota monax, woodchuck), AF177031(Rattus norvegicus, rat), and M86672, S82419 (Mus musculus, mouse). Suchunique subsequences can be determined by aligning any of SEQ ID NO:1 toSEQ ID NO: 7, or SEQ ID NO:16 to SEQ ID NO:25 against the complete setof nucleic acids corresponding to GenBank accession numbers of known p40or p35 sequences, such as those listed above or other similar p40 or p35sequences presented in GenBank. Alignment can be performed using theBLAST algorithm set to default parameters. Any unique subsequence isuseful, e.g., as a probe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from: SEQ ID NO:8 to SEQ ID NO:14,and SEQ ID NO: 26 to SEQ ID NO:35. Here, the unique subsequence isunique as compared to a an amino acid subsequence of a known p40 or p35polypeptide including, e.g., an amino acid subsequence of a polypeptideencoded by a known p40 or p35 nucleic acid corresponding to any ofGenBank accession numbers listed above, or other similar p40 or p35nucleic acid or polypeptide sequences presented in GenBank. Here again,the polypeptide is aligned against the complete set of known p40 or p35polypeptide sequences, such as those polypeptides encoded by the nucleicacids corresponding to the GenBank accession numbers listed above, orother similar p40 or p35 nucleic acid or polypeptide sequences presentedin GenBank (referred to as the “control peptides”; note that where thesequence corresponds to a non-translated sequence such as a pseudo gene,the corresponding polypeptide is generated simply by in silicotranslation of the nucleic acid sequence into an amino acid sequence,where the reading frame is selected to correspond to the reading frameof homologous p40 or p35 nucleic acids).

The invention also provides a target nucleic acid which hybridizes underat least stringent or highly stringent conditions (or conditions ofgreater stringency) conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from: SEQ ID NO:8to SEQ ID NO:14, and SEQ ID NO:26 to SEQ ID NO:35, wherein the uniquesubsequence is unique as compared to an amino acid subsequence of aknown p40 or p35 polypeptide sequence shown in GenBank or to apolypeptide corresponding to any of the control polypeptides. Uniquesequences are determined as noted above.

In one example, the stringent conditions are selected such that aperfectly complementary oligonucleotide to the coding oligonucleotidehybridizes to the coding oligonucleotide with at least about a 2.5×-10×higher, preferably at least about a 5-10× higher signal to noise ratiothan for hybridization of the perfectly complementary oligonucleotide toa control nucleic acid corresponding to any of the control polypeptides.Conditions can be selected such that higher ratios of signal to noiseare observed in the particular assay which is used, e.g., about 15×,20×, 30×, 50× or more. In this example, the target nucleic acidhybridizes to the unique coding oligonucleotide with at least a 2×higher signal to noise ratio as compared to hybridization of the controlnucleic acid to the coding oligonucleotide. Again, higher signal tonoise ratios can be selected, e.g., about 2.5×, 5×, 10×, 20×, 30×, 50×or more. The particular signal will depend on the label used in therelevant assay, e.g., a fluorescent label, a colorimetric label, aradioactive label, or the like.

Substrates and Formats for Sequence Recombination

The polynucleotides of the invention are useful as substrates for avariety of diversity generation, recombination and recursive sequencerecombination (e.g., DNA shuffling) reactions, as well as otherdiversity generating techniques, including mutagenesis techniques andstandard cloning methods as set forth in, e.g., Ausubel, Berger andSambrook, supra, i.e., to produce additional modified p40 polypeptidesor modified p35 polypeptides with desired properties. Based on thescreening or selection protocols employed, recombinant, e.g., shuffled,modified p40 or modified p35 polypeptides can be generated and isolatedthat confer a variety of desirable characteristics, e.g., enhancedT-cell proliferative activity, enhanced interferon-gamma inductiveactivity, enhanced T_(H)1 cell differentiation activity, reducedtoxicity, reduced immunogenicity, etc.

A variety of diversity generating protocols, including nucleic acidshuffling protocols, are available and fully described in the art. Theprocedures can be used separately, and/or in combination to produce oneor more variants of a nucleic acid or set of nucleic acids, as wellvariants of encoded proteins. Individually and collectively, theseprocedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel, or in series toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids that encode proteins with orwhich confer desirable properties. Following diversification by one ormore of the methods herein, or otherwise available to one of skill, anynucleic acids that are produced can be selected for a desired activityor property, e.g., enhanced T-cell proliferative activity, enhancedinterferon-gamma induction activity, enhanced T_(H)1 celldifferentiation activity, reduced toxicity, reduced immunogenicity, etc.Methods for determining nucleic acids having enhanced T-cellproliferative activity, enhanced interferon-gamma inductive activity,enhanced T_(H)1 cell differentiation activity, reduced toxicity, reducedimmunogenicity, etc., include those described herein. This can includeidentifying any activity that can be detected, for example, in anautomated or automatable format, by any of the assays in the art. Avariety of related (or even unrelated) properties can be evaluated inserial or in parallel at the discretion of the practitioner.

The following publications describe a variety of diversity generatingprocedures, including recursive sequence recombination procedures (alsotermed simply “recursive recombination), and/or methods for generatingmodified nucleic acid sequences for use in the procedures and methods ofthe present invention include the following publications and thereferences cited therein: Soong, N. W. et al. (2000) “Molecular Breedingof Viruses,” Nature Genetics 25:436-439; Stemmer, W. et al. (1999)“Molecular breeding of viruses for targeting and other clinicalproperties,” Tumor Targeting 4:1-4; Ness et al. (1999) “DNA Shuffling ofsubgenomic sequences of subtilisin,” Nature Biotechnology 17:893-896;Chang et al. (1999) “Evolution of a cytokine using DNA familyshuffling,” Nature Biotechnology 17:793-797; Minshull and Stemmer (1999)“Protein evolution by molecular breeding,” Current Opinion in ChemicalBiology 3:284-290; Christians et al. (1999) “Directed evolution ofthymidine kinase for AZT phosphorylation using DNA family shuffling,”Nature Biotechnology 17:259-264; Crameri et al. (1998) “DNA shuffling ofa family of genes from diverse species accelerates directed evolution,”Nature 391:288-291; Crameri et al. (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang et al. (1997) “Directed evolution of an effectivefucosidase from a galactosidase by DNA shuffling and screening,” Proc.Nat'l Acad. Sci. USA 94:4504-4509; Patten et al. (1997) “Applications ofDNA Shuffling to Pharmaceuticals and Vaccines,” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling,” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling,” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer,’” J. Mol. Biol. 255:373-386; Stemmer (1996) “Sexual PCR andAssembly PCR” In: The Encyclopedia of Molecular Biology, VCH Publishers,New York. pp. 447-457; Crameri and Stemmer (1995) “Combinatorialmultiple cassette mutagenesis creates all the permutations of mutant andwildtype cassettes,” BioTechniques 18:194-195; Stemmer et al. (1995)“Single-step assembly of a gene and entire plasmid form large numbers ofoligodeoxy-ribonucleotides” Gene 164:49-53; Stemmer (1995) “TheEvolution of Molecular Computation,” Science 270:1510; Stemmer (1995)“Searching Sequence Space,” Bio/Technology 13:549-553; Stemmer (1994)“Rapid evolution of a protein in vitro by DNA shuffling,” Nature370:389-391; and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution,” Proc.Nat'l Acad. Sci. USA 91:10747-10751.

Additional details regarding DNA shuffling and other diversitygenerating methods can be found in the following U.S. patents, PCTpublications, and EP publications: U.S. Pat. No. 5,605,793 to Stemmer(Feb. 25, 1997), “Methods for In vitro Recombination;” U.S. Pat. No.5,811,238 to Stemmer et al. (Sep. 22, 1998) “Methods for GeneratingPolynucleotides having Desired Characteristics by Iterative Selectionand Recombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S.Pat. No. 5,834,252 to Stemmer (Nov. 10, 1998) “End-ComplementaryPolymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull (Nov. 17,1998), “Methods and Compositions for Cellular and MetabolicEngineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by RandomFragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz,“End Complementary Polymerase Chain Reaction;” WO 97/20078 by Stemmerand Crameri “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” WO 97/35966by Minshull and Stemmer, “Methods and Compositions for Cellular andMetabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting ofGenetic Vaccine Vectors;” WO 99/41383 by Punnonen et al., “AntigenLibrary Immunization;” WO 99/41369 by Punnonen et al., “Genetic VaccineVector Engineering;” WO 99/41368 by Punnonen et al., “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by Del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” EP 0946755 by Patten andStemmer, “Methods and Compositions for Polypeptide Engineering;” and WO98/13487 by Stemmer et al., “Methods for Optimization of Gene Therapy byRecursive Sequence Shuffling and Selection;” WO 00/00632, “Methods forGenerating Highly Diverse Libraries,” WO 00/09679, “Methods forObtaining in vitro Recombined Polynucleotide Sequence Banks andResulting Sequences,” WO 98/42832 by Arnold et al., “Recombination ofPolynucleotide Sequences Using Random or Defined Primers,” WO 99/29902by Arnold et al., “Method for Creating Polynucleotide and PolypeptideSequences,” WO 98/41653 by Vind, “An in vitro Method for Construction ofa DNA Library,” WO 98/41622 by Borchert et al., “Method for Constructinga Library Using DNA Shuffling,” and WO 98/42727 by Pati and Zarling,“Sequence Alterations using Homologous Recombination.”

Certain U.S. applications provide additional details regarding DNAshuffling and related techniques, as well as other diversity generatingmethods, including “SHUFFLING OF CODON ALTERED GENES” by Patten et al.filed Sep. 29, 1998 (U.S. Ser. No. 60/102,362), Jan. 29, 1999 (U.S. Ser.No. 60/117,729), and Sep. 28, 1999 (U.S. Ser. No. 09/407,800);“EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCERECOMBINATION”, by Del Cardayre et al. filed Jul. 15, 1998 (U.S. Ser.No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Feb. 5, 1999 (U.S. Ser. No. 60/118,813), Jun. 24, 1999 (U.S. Ser.No. 60/141,049), and Sep. 28, 1999 (U.S. Ser. No. 09/408,392); “USE OFCODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welchet al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393); “METHODS FORMAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov and Stemmer, filed Feb. 5, 1999 (U.S. Ser.No. 60/118,854) and Oct. 12, 1999 (U.S. Ser. No. 09/416,375);RECOMBINATION OF INSERTION MODIFIED NUCLEIC ACIDS by Patten et al.,filed Mar. 5, 1999 (U.S. Ser. No. 60/122,943), Jul. 2, 1999 (U.S. Ser.No. 60/142,299), Nov. 10, 1999 (U.S. Ser. No. 60/164,618), and Nov. 10,1999 (U.S. Ser. No. 60/164,617); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, U.S. Ser. No. 60/186,482 filed Mar. 2, 2000.

As a review of the foregoing publications, patents, published foreignapplications and U.S. patent applications reveals, diversity generationmethods, such as shuffling (or recursive sequence recombination) ofnucleic acids to provide new nucleic acids with desired properties canbe carried out by a number of established methods. Any of these methodscan be adapted to the present invention to evolve the p40 and p35polypeptides discussed herein to produce new modified p40 or modifiedp35 polypeptides with new or improved properties. Both the methods ofmaking such polypeptides and the polypeptides (e.g., modified p40polypeptides and modified p35 polypeptides) produced by these methodsare a feature of the invention. In brief, several different generalclasses of sequence modification methods, such as recombination, areapplicable to the present invention and set forth, e.g., in thereferences above. First, nucleic acids can be recombined in vitro by anyof a variety of techniques discussed in the references above, includinge.g., DNAse digestion of nucleic acids to be recombined followed byligation and/or PCR reassembly of the nucleic acids. Second, nucleicacids can be recursively recombined in vivo or ex vivo, e.g., byallowing recombination to occur between nucleic acids in cells. Third,whole genome recombination methods can be used in which whole genomes ofcells or other organisms are recombined, optionally including spiking ofthe genomic recombination mixtures with desired library components(e.g., genes corresponding to the pathways of the present invention).Fourth, synthetic recombination methods can be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Fifth, in silico methodsof recombination can be effected in which genetic algorithms are used ina computer to recombine sequence strings which correspond to homologous(or even non-homologous) nucleic acids. The resulting recombinedsequence strings are optionally converted into nucleic acids bysynthesis of nucleic acids which correspond to the recombined sequences,e.g., in concert with oligonucleotide synthesis/gene reassemblytechniques. Any of the preceding general recombination formats can bepracticed in a reiterative fashion to generate a more diverse set ofrecombinant nucleic acids. Sixth, methods of accessing naturaldiversity, e.g., by hybridization of diverse nucleic acids or nucleicacid fragments to single-stranded templates, followed by polymerizationand/or ligation to regenerate full-length sequences, optionally followedby degradation of the templates and recovery of the resulting modifiednucleic acids can be used. above references provide these and otherbasic recombination formats as well as many modifications of theseformats. Regardless of the format which is used, the nucleic acids ofthe invention can be recombined (with each other, or with related (oreven unrelated) nucleic acids to produce a diverse set of recombinantnucleic acids, including e.g., homologous nucleic acids. In general, thesequence recombination techniques described herein provide particularadvantages in that they provide for recombination between the nucleicacids of SEQ ID NO:1 to SEQ ID NO:7, or SEQ ID NO:16 to SEQ ID NO:25, orfragments or variants thereof, in any available format, therebyproviding a very fast way of exploring the manner in which differentcombinations of sequences can affect a desired result.

Following recombination, any nucleic acids which are produced can bescreened or selected for a desired activity. In the context of thepresent invention, this can include testing for and identifying anyactivity that can be detected, e.g., in an automatable format, by anyassay known in the art. In addition, useful properties such as lowimmunogenicity, increased half-life, improved solubility, oralavailability, or the like can also be selected for. A variety of p40and/or p35 related (or even unrelated) properties can be assayed for,using any available assay.

DNA mutagenesis and recursive recombination provide a robust, widelyapplicable, means of generating diversity useful for the engineering ofproteins, pathways, cells and organisms with improved characteristics.In addition to the basic formats described above, it is sometimesdesirable to combine shuffling methodologies with other techniques forgenerating diversity. In conjunction with (or separately from) shufflingmethods, a variety of diversity generation methods can be practiced andthe results (i.e., diverse populations of nucleic acids) screened for inthe systems of the invention. Additional diversity can be introduced bymethods which result in the alteration of individual nucleotides orgroups of contiguous or non-contiguous nucleotides, i.e., mutagenesismethods. Many mutagenesis methods are found in the above-citedreferences; additional details regarding mutagenesis methods can befound in the references listed below.

Mutagenesis methods of generating diversity include, for example,recombination (PCT/US98/05223; Publ. No. WO98/42727); site-directedmutagenesis (Ling et al. (1997) “Approaches to DNA mutagenesis: anoverview,” Anal. Biochem. 254(2):157-178; Dale et al. (1996)“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod,” Methods Mol. Biol. 57:369-374; Smith (1985) “In vitromutagenesis,” Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985)“Strategies and applications of in vitro mutagenesis,” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis,” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis,” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection,” Proc. Nat'lAcad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection,” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities,” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100:468-500(1983); Methods in Enzymol. 154:329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment,” Nucleic Acids Res. 10:6487-6500; Zoller & Smith(1983) “Oligonucleotide-directed mutagenesis of DNA fragments clonedinto M13 vectors,” Methods in Enzymol. 100:468-500; and Zoller & Smith(1987) “Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template,” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA,” Nucl. Acids Res. 13:8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA,” Nucl.Acids Res. 13:8765-8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis,” Nucl.Acids Res. 14:9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis,” Nucl.Acids Res. 16:791-802; and Sayers et al. (1988) “Strand specificcleavage of phosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide,” Nucl. Acids Res.16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984)“The gapped duplex DNA approach to oligonucleotide-directed mutationconstruction,” Nucl. Acids Res. 12:9441-9456; Kramer & Fritz (1987)“Oligonucleotide-directed construction of mutations via gapped duplexDNA,” Methods in Enzymol. 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations,” Nucl. Acids Res.16:7207; and Fritz et al. (1988) “Oligonucleotide-directed constructionof mutations: a gapped duplex DNA procedure without enzymatic reactionsin vitro,” Nucl. Acids Res. 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer et al.(1984) “Point Mismatch Repair,” Cell 38:879-887), mutagenesis usingrepair-deficient host strains (Carter et al. (1985) “Improvedoligonucleotide site-directed mutagenesis using M13 vectors,” Nucl.Acids Res. 13:4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors,” Methods inEnzymol. 154:382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff(1986) “Use of oligonucleotides to generate large deletions,” Nucl.Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin,” Phil. Trans. R. Soc. Lond. A 317:415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein,” Science 223:1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducing),” Nucl. Acids Res. 14:6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites,” Gene 34:315-323; and Grundström etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis,” Nucl. Acids Res. 13:3305-3316),double-strand break repair (Mandecki (1986) “Oligonucleotide-directeddouble-strand break repair in plasmids of Escherichia coli: a method forsite-specific mutagenesis,” Proc. Nat'l Acad. Sci. USA, 83:7177-7181).Additional details on many of the above methods can be found in MethodsEnzymol., Vol. 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Random or semi-random mutagenesis using doped or degenerateoligonucleotides (Arkin and Youvan (1992) “Optimizing nucleotidemixtures to encode specific subsets of amino acids for semi-randommutagenesis,” Biotechnology 10:297-300; Reidhaar-Olson et al. (1991)“Random mutagenesis of protein sequences using oligonucleotidecassettes,” Methods in Enzymol. 208:564-86; Lim and Sauer (1991) “Therole of internal packing interactions in determining the structure andstability of a protein,” J. Mol. Biol. 219:359-76; Breyer and Sauer(1989) “Mutational analysis of the fine specificity of binding ofmonoclonal antibody 51F to lambda repressor,” J. Biol. Chem.264:13355-60); “Walk-Through Mutagenesis” (Crea, R.; U.S. Pat. Nos.5,830,650 and 5,798,208, and EP Patent 0527809 B1) may also be employedto generate diversity.

In one aspect of the present invention, error-prone PCR can be used togenerate nucleic acid variants. Using this technique, PCR is performedunder conditions where the copying fidelity of the DNA polymerase islow, such that a high rate of point mutations is obtained along theentire length of the PCR product. Examples of such techniques are foundin the references above and, e.g., in Leung et al. (1989) Technique1:11-15 and Caldwell et al. (1992) PCR Methods Applic. 2:28-33.Similarly, assembly PCR can be used, in a process which involves theassembly of a PCR product from a mixture of small DNA fragments. A largenumber of different PCR reactions can occur in parallel in the samevial, with the products of one reaction priming the products of anotherreaction. Sexual PCR mutagenesis can be used in which homologousrecombination occurs between DNA molecules of different but related DNAsequence in vitro, by random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. This process is described in the referencesabove, e.g., in Stemmer (1994) Proc. Nat'l Acad. Sci. USA91:10747-10751. Recursive ensemble mutagenesis can be used in which analgorithm for protein mutagenesis is used to produce diverse populationsof phenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Nat'l Acad. Sci. USA89:7811-7815.

As noted, oligonucleotide directed mutagenesis can be used in a processwhich allows for the generation of site-specific mutations in anynucleic acid sequence of interest. Examples of such techniques are foundin the references above and, e.g., in Reidhaar-Olson et al. (1988)Science, 241:53-57. Similarly, cassette mutagenesis can be used in aprocess which replaces a small region of a double stranded DNA moleculewith a synthetic oligonucleotide cassette that differs from the nativesequence. The oligonucleotide can contain, e.g., completely and/orpartially randomized native sequence(s).

In vivo (or ex vivo) mutagenesis can be used in a process of generatingrandom mutations in any cloned DNA of interest which involves thepropagation of the DNA, e.g., in a strain of E. coli that carriesmutations in one or more of the DNA repair pathways. These “mutator”strains have a higher random mutation rate than that of a wild-typeparent. Propagating the DNA in one of these strains will eventuallygenerate random mutations within the DNA.

Exponential ensemble mutagenesis can be used for generatingcombinatorial libraries with a high percentage of unique and functionalmutants, where small groups of residues are randomized in parallel toidentify, at each altered position, amino acids which lead to functionalproteins. Examples of such procedures are found in Delegrave & Youvan(1993) Biotechnology Research 11:1548-1552. Similarly, random andsite-directed mutagenesis can be used. Examples of such procedures arefound in Arnold (1993) Current Opinion in Biotechnology 4:450-455.

Kits for mutagenesis, library construction, and other diversitygeneration methods are also commercially available. For example, kitsare available from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit; and Chameleon™ double-stranded, site-directedmutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod described above), Boehringer Mannheim Corp., ClontechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), NewEngland Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc (e.g., using the Ecksteinmethod above), and Anglian Biotechnology Ltd (e.g., using theCarter/Winter method above).

Any of the described shuffling or mutagenesis techniques can be used inconjunction with procedures which introduce additional diversity into agenome, e.g., a bacterial, fungal, animal or plant genome. For example,in addition to the methods above, techniques have been proposed whichproduce chimeric nucleic acid multimers suitable for transformation intoa variety of species (see, e.g., Schellenberger U.S. Pat. No. 5,756,316and the references above). When such chimeric multimers consist of genesthat are divergent with respect to one another (e.g., derived fromnatural diversity or through application of site directed mutagenesis,error prone PCR, passage through mutagenic bacterial strains, and thelike), are transformed into a suitable host, this provides a source ofnucleic acid diversity for DNA diversification.

Chimeric multimers transformed into host species are suitable assubstrates for in vivo (or ex vivo) shuffling protocols. Alternatively,a multiplicity of polynucleotides sharing regions of partial sequencesimilarity or homology can be transformed into a host species andrecombined in vivo (or ex vivo) by the host cell. Subsequent rounds ofcell division can be used to generate libraries, members of which,comprise a single, homogenous population of monomeric or pooled nucleicacid. Alternatively, the monomeric nucleic acid can be recovered bystandard techniques and recursively recombined in any of the describedshuffling formats.

Chain termination methods of diversity generation have also beenproposed (see, e.g., U.S. Pat. No. 5,965,408 and the references above).In this approach, double stranded DNAs corresponding to one or moregenes sharing regions of sequence similarity or homology are combinedand denature, in the presence or absence of primers specific for thegene. The single stranded polynucleotides are then annealed andincubated in the presence of a polymerase and a chain terminatingreagent (e.g., ultraviolet, gamma or X-ray irradiation; ethidium bromideor other intercalators; DNA binding proteins, such as single strandbinding proteins, transcription activating factors, or histones;polycyclic aromatic hydrocarbons; trivalent chromium or a trivalentchromium salt; or abbreviated polymerization mediated by rapidthermocycling; and the like), resulting in the production of partialduplex molecules. The partial duplex molecules, e.g., containingpartially extended chains, are then denatured and reannealed insubsequent rounds of replication or partial replication resulting inpolynucleotides which share varying degrees of sequence similarity orhomology and which are chimeric with respect to the starting populationof DNA molecules. Optionally, the products or partial pools of theproducts can be amplified at one or more stages in the process.Polynucleotides produced by a chain termination method, such asdescribed above are suitable substrates for diversity generation methods(e.g., RSR, DNA shuffling) according to any of the described formats.

Diversity can be further increased by using methods which are nothomology based with DNA shuffling (which, as set forth in the abovepublications and applications can be homology or non-homology based,depending on the precise format). For example, incremental truncationfor the creation of hybrid enzymes (ITCHY) described in Ostermeier etal. (1999) “A combinatorial approach to hybrid enzymes independent ofDNA homology,” Nature Biotechnol. 17:1205, can be used to generate aninitial recombinant library which serves as a substrate for one or morerounds of in vitro, ex vivo, or in vivo diversity generation methods(e.g., RSR or shuffling methods).

Methods for generating multispecies expression libraries have beendescribed (e.g., U.S. Pat. Nos. 5,783,431; 5,824,485 and the referencesabove) and their use to identify protein activities of interest has beenproposed (U.S. Pat. No. 5,958,672 and the references above).Multispecies expression libraries are, in general, libraries comprisingcDNA or genomic sequences from a plurality of species or strains,operably linked to appropriate regulatory sequences, in an expressioncassette. The cDNA and/or genomic sequences are optionally randomlyconcatenated to further enhance diversity. The vector can be a shuttlevector suitable for transformation and expression in more than onespecies of host organism, e.g., bacterial species, eukaryotic cells. Insome cases, the library is biased by preselecting sequences which encodea protein of interest, or which hybridize to a nucleic acid of interest.Any such libraries can be provided as substrates for any of the methodsherein described.

In some applications, it is desirable to preselect or prescreenlibraries (e.g., an amplified library, a genomic library, a cDNAlibrary, a normalized library, etc.) or other substrate nucleic acidsprior to shuffling, or to otherwise bias the substrates towards nucleicacids that encode functional products (shuffling procedures can also,independently have these effects). For example, in the case of antibodyengineering, it is possible to bias the shuffling process towardantibodies with functional antigen binding sites by taking advantage ofin vivo (or ex vivo or in vitro) recombination events prior to diversitygeneration (e.g., DNA shuffling) by any described method. For example,recombined CDRs derived from B cell cDNA libraries can be amplified andassembled into framework regions (e.g., Jirholt et al. (1998)“Exploiting sequence space: shuffling in vivo formed complementaritydetermining regions into a master framework,” Gene 215:471) prior todiversity generation (e.g., DNA shuffling) according to any of themethods described herein.

Libraries can be biased towards nucleic acids which encode proteins withdesirable activities (e.g., binding affinities, enzymatic activities,proliferative activities, cell differentiation activities, ability toinduce an immune response, adjuvant properties, etc.). For example,after identifying a clone from a library which exhibits a specifiedactivity, the clone can be mutagenized using any known method forintroducing DNA alterations, including, but not restricted to, DNAshuffling or another form of recursive sequence recombination ordiversity generation. A library comprising the mutagenized clones isthen screened for a desired activity, which can be the same as ordifferent from the initially specified activity. An example of such aprocedure is proposed in U.S. Pat. No. 5,939,250. Desired activities canbe identified by any method known in the art. For example, WO 99/10539proposes that gene libraries can be screened by combining extracts fromthe gene library with components obtained from metabolically rich cellsand identifying combinations which exhibit the desired activity. It hasalso been proposed (e.g., WO 98/58085) that clones with desiredactivities can be identified by inserting bioactive substrates intosamples of the library, and detecting bioactive fluorescencecorresponding to the product of a desired activity using a fluorescentanalyzer, e.g., a flow cytometry device, a CCD, a fluorometer, or aspectrophotometer.

Libraries can also be biased towards nucleic acids which have specifiedcharacteristics, e.g., hybridization to a selected nucleic acid probe.For example, application WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences in the following manner.Single stranded DNA molecules from a population of genomic DNA arehybridized to a ligand-conjugated probe. The genomic DNA can be derivedfrom either a cultivated or uncultivated microorganism, or from anenvironmental sample. Alternatively, the genomic DNA can be derived froma multicellular organism, or a tissue derived therefrom.

Second strand synthesis can be conducted directly from the hybridizationprobe used in the capture, with or without prior release from thecapture medium or by a wide variety of other strategies known in theart. Alternatively, the isolated single-stranded genomic DNA populationcan be fragmented without further cloning and used directly in ashuffling-based gene reassembly process. In one such method the fragmentpopulation derived the genomic library(ies) is annealed with partial,or, often approximately full length ssDNA or RNA corresponding to theopposite strand. Assembly of complex chimeric genes from this populationis the mediated by nuclease-base removal of non-hybridizing fragmentends, polymerization to fill gaps between such fragments and subsequentsingle stranded ligation. The parental strand can be removed bydigestion (if RNA or uracil-containing), magnetic separation underdenaturing conditions (if labeled in a manner conducive to suchseparation) and other available separation/purification methods.Alternatively, the parental strand is optionally co-purified with thechimeric strands and removed during subsequent screening and processingsteps. As set forth in “Single-stranded nucleic acid template-mediatedrecombination and nucleic acid fragment isolation” by Affholter (U.S.Ser. No. 60/186,482, filed Mar. 2, 2000) and WO 98/27230, “Methods andCompositions for Polypeptide Engineering” by Patten and Stemmer,shuffling using single-stranded templates and nucleic acids of interestwhich bind to a portion of the template can also be performed.

In one approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for any of the shuffling reactions described herein.

“Non-Stochastic” methods of generating nucleic acids and polypeptidesare alleged in Short, J. “Non-Stochastic Generation of Genetic Vaccinesand Enzymes,” WO 00/46344. These methods, including the proposednon-stochastic polynucleotide reassembly and gene site saturationmutagenesis and synthetic ligation polynucleotide reassembly methodsoutlined therein, can be applied to the present invention as well.

It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods.

A recombinant nucleic acid produced by recursively recombining one ormore polynucleotides of the invention with one or more additionalnucleic acids also forms a part of the invention. The one or moreadditional nucleic acids may include another polynucleotide of theinvention; optionally, alternatively, or in addition, the one or moreadditional nucleic acids can include, e.g., a nucleic acid encoding anaturally-occurring p40 polypeptide or p35 polypeptide, or a subsequencethereof, or any homologous p40 polypeptide or p35 polypeptide sequenceor subsequence thereof (e.g., a p40 or p35 sequence as found in GenBankor other available literature), or, e.g., any other homologous ornon-homologous nucleic acid (certain recombination formats noted above,notably those performed synthetically or in silico, do not requirehomology for recombination).

The recombining steps may be performed in vivo, ex vivo, in vitro, inplanta, or in silico as described in more detail in the referencesabove. Also included in the invention is a cell containing any resultingrecombinant nucleic acid, nucleic acid libraries produced by diversitygeneration, recombination, or recursive sequence recombination of thenucleic acids set forth herein, and populations of cells, vectors,viruses, plasmids or the like comprising the library or comprising anyrecombinant nucleic acid resulting from diversity generation orrecombination (or recursive sequence recombination) of a nucleic acid asset forth herein with another such nucleic acid, or an additionalnucleic acid. Corresponding sequence strings in a database present in acomputer system or computer readable medium are a feature of theinvention.

Other Polynucleotide Compositions

The invention also includes compositions comprising two or morepolynucleotides of the invention (e.g., as substrates forrecombination). The composition can comprise a library of recombinantnucleic acids, where the library contains at least 2, 3, 5, 10, 20, or50 or more nucleic acids. The nucleic acids are optionally cloned intoexpression vectors, providing expression libraries.

The invention also includes compositions produced by digesting one ormore polynucleotide of the invention with a restriction endonuclease, anRNAse, or a DNAse (e.g., as is performed in certain of the recombinationformats noted above); and compositions produced by fragmenting orshearing one or more polynucleotide of the invention by radiation,chemical, or mechanical means (e.g., sonication, vortexing, and thelike), which can also be used to provide substrates for recombination inthe methods above. Similarly, compositions comprising sets ofoligonucleotides corresponding to more than one nucleic acid of theinvention are useful as recombination substrates and are a feature ofthe invention. For convenience, these fragmented, sheared, oroligonucleotide synthesized mixtures are referred to as fragmentednucleic acid sets.

Also included in the invention are compositions produced by incubatingone or more of the fragmented nucleic acid sets in the presence ofribonucleotide- or deoxyribonucelotide triphosphates and a nucleic acidpolymerase. This resulting composition forms a recombination mixture formany of the recombination formats noted above. The nucleic acidpolymerase may be an RNA polymerase, a DNA polymerase, or anRNA-directed DNA polymerase (e.g., a “reverse transcriptase”); thepolymerase can be, e.g., a thermostable DNA polymerase (such as, VENT,TAQ, or the like).

Polypeptides of the Invention

The invention provides isolated or recombinant polypeptides,collectively referred to herein as “modified cytokine polypeptides,”“modified p35 polypeptides,”, “modified p40 polypeptides,” or simply“polypeptides of the invention.”

Modified p40 Polypeptides

An isolated or recombinant modified p40 polypeptide of the inventionincludes a polypeptide comprising a mature polypeptide region of asequence selected from SEQ ID NO:8 to SEQ ID NO: 14, conservativelymodified variations thereof, and fragments thereof having proliferativeactivity or interferon-gamma induction activity in a T-cell based assay(such as, e.g., a human T-cell based assay), in the presence of a p35polypeptide (such as, e.g., a wt-p35 polypeptide or a modified p35polypeptide). An alignment of exemplary modified p40 polypeptidesequences according to the invention is provided in FIG. 1. Alignment ofthe polypeptide sequences of the invention to each other or to sequencesof known or naturally-occurring p40 polypeptides is readily performed byone of ordinary skill in the art using publicly available databases andalignment programs. Sequences of known, naturally-occurring p40polypeptides from human and non-human sources are readily available froma variety of sources, such as GenBank or SWISSPROT.

The modified p40 polypeptide of the invention comprises at least onemodification at a position equivalent to that in the amino acid sequenceof a human p40 polypeptide (SEQ ID NO:15). The modification can include:

(a) a substitution of the specified amino acid for a different aminoacid at one or more equivalent position selected from Leu62, Ser71,Gln78, His99, Thr127, Arg130, Lys185, Glu186, Tyr187, Glu188, Ser190,Asp196, Met211, Val289, Ser305, Ser307, Arg309, and Gln311;

(b) a deletion of one or more of equivalent amino acids Arg181 to Asn184inclusive, or a substitution of said equivalent amino acids for one ormore of the amino acids Ser-(Leu or Met)-(Glu or Asp)-His-Arg; and

(c) a deletion of one or more of equivalent amino acids Asp287 andArg288.

A modified p40 polypeptide of the invention can optionally contain atleast two of modifications (a), (b) or (c). For purposes of clarity, thenumbering of amino acid positions corresponds to that of a human p40(SEQ ID NO:15); however, it is understood that such modification may bemade in an equivalent position of any p40 polypeptide.

An “equivalent position”, or a “position equivalent to”, refers to aposition related by sequence homology to a specified position of thecomparison sequence (in this example, SEQ ID NO:15). For example, inFIG. 1 the amino acid sequence of a human p40 (SEQ ID NO:15) andexemplary modified p40 polypeptide sequences of the invention (SEQ IDNO:8 to SEQ ID NO:14) are aligned to provide the maximum amount ofhomology between the sequences. A comparison of these sequences showsthat there are a number of conserved residues contained in eachsequence. As can be seen, there are also a number of insertions anddeletions in the exemplary modified p40 sequences as compared to thehuman p40 sequence.

Thus, for example, inspection of FIG. 1 shows that the amino acidposition equivalent to amino acid 185 in the human p40 sequence SEQ IDNO:15 (i.e., the equivalent position to amino acid 185), is 181 in themodified p40 polypeptide sequence SEQ ID NO:8, owing to the deletion ofArg-Gly-Asp-Asn at position 181 to 184 inclusive, and is 186 in themodified p40 polypeptide sequence SEQ ID NO:9, owing to the substitutionof Arg-Gly-Asp-Asn for the amino acids Ser-(Leu or Met)-(Glu orAsp)-His-Arg at that same position. Likewise, the amino acid positionequivalent to amino acid 289 in the human p40 sequence (SEQ ID NO:15) is285 in the modified p40 sequence SEQ ID NO:8, and is 288 in the modifiedp40 sequence SEQ ID NO:9, owing to a deletion of amino acids Asp-Arg atequivalent positions 287 and 288 of SEQ ID NO:15.

In one embodiment, a modified p40 polypeptide of the invention comprisesat least one substitution at a position equivalent to that of the aminoacid sequence of human p40 polypeptide (SEQ ID NO:15), selected from thegroup consisting of: Leu62Ser; Ser71Thr; Gln78His; His99(Arg or Gln);Thr127(Ser or Ile); Arg130Lys; Lys185Glu; Glu186Tyr; Tyr187(Lys or Asn);Glu188Lys; Ser190(Arg or Thr); Asp196Gly; Met211Val; Val289(Ile or Leu);Ser305Lys; Ser307Arg; Arg309Gln; and Gln311Arg. In other embodiments, amodified p40 polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, or 17 of the above substitutions. In anotherembodiment, a modified p40 polypeptide of the invention comprises all ofthe above substitutions.

A modified p40 polypeptide of the invention optionally further comprisesat least one of the following substitutions in an position equivalent tothat of SEQ ID NO:15: Cys2His; His3Pro; Ile8Val; Phe15Leu; Val21Met;Lys27Glu; Asp29Asn; Asp40Asn; Met45Thr; Thr49Ala; Glu67Gly, His91Arg;Glu95(Ala or Thr), Val96Ala; Glu122Lys; Asn125Ala; Asn135Asp; Arg139His;Thr147Ala; Thr153Lys; Ser155Thr; Ser163Thr; Gln166(Arg or His),Ala172Thr; Ala173Val; Thr174Leu; Ala177Glu; Glu178Asp; Arg179Leu;Val180Gly; Ala201Ser; Val212Leu; Asp213Glu; Val215Ile; Ser226Arg;Lys244Arg; Gln251His; Val254Ile; Ser255Asn; Glu257Gly; Thr264(Ala orIle); Thr272Met; Cys274Gly; Val275Ile; Lys280Arg; Ser281Asn; Lys285Asp;Lys286Arg; Phe290Ser; Thr291(Met or Val); Lys293Gln; Thr297Lys;Ile299(Thr or Val); Arg301His; Asn303Asp; Ser318Phe; Glu321Asp;Pro326Ser; Cys327Leu; and Ser328(Gly or Gln).

The modified p40 polypeptide sequence may be a modification of anaturally-occurring p40 polypeptide sequence of a mammal (e.g., human, anon-human primate, a ruminant, or a rodent), preferably a primate, morepreferably human. The naturally-occurring p40 polypeptide sequence maybe one encoded by one of the following nucleic acid sequences havingGenBank accession number: M65272, M65290 (human); U19841 (Macacamulatta, rhesus monkey); U19834 (Cercocebus torquatus, sooty mangabey);Y11129 (Equus caballus, horse); U83184, Y07762, AF054607 (Felis catus,cat); U49100, AF091134 (Canis familiaris, dog); U57752, U10160 (Cervuselaphus, red deer); AF007576 (Capra hircus, goat); AF004024 (Ovis aries,sheep); U11815 (Bos taurus, cow); U08317 (Sus scrofa, pig); X97019,AF082494 (Marmota monax, woodchuck); AF133197, U16674 (Rattusnorvegicus, rat); M86671, S82426 (Mus musculus, mouse); AF097507 (Caviaporcellus, guinea pig); and AF046211 (Mesocricetus auratus, goldenhamster). The naturally-occurring p40 polypeptide sequence is morepreferably a primate sequence, still more preferably a human sequence,most preferably a mature human p40 sequence comprising amino acidresidues 23 to 328 of SEQ ID NO:15.

The invention also includes an isolated or recombinant polypeptidecomprising an amino acid sequence containing at least 10 contiguousamino acid residues of any one of SEQ ID NOS:8-14. The polypeptidetypically includes at least one amino acid substitution, at anequivalent position to that of SEQ ID NO:15, selected from: Leu62Ser;Ser71Thr; Gln78His; His99(Arg or Gln); Thr127(Ser or Ile); Arg130Lys;Lys185Glu; Glu186Tyr; Tyr187(Lys or Asn); Glu188Lys; Ser190(Arg or Thr);Asp196Gly; Met211Val; Val289(Ile or Leu); Ser305Lys; Ser307Arg;Arg309Gln; and Gln311Arg.

The modified p40 polypeptide may optionally comprise at least onesubstitution selected from: Cys2His; His3Pro; Ile8Val; Phe15Leu;Val21Met; Lys27Glu; Asp29Asn; Asp40Asn; Met45Thr; Thr49Ala; Glu67Gly,His91Arg; Glu95(Ala or Thr), Val96Ala; Glu122Lys; Asn125Ala; Asn135Asp;Arg139His; Thr147Ala; Thr153Lys; Ser155Thr; Ser163Thr; Gln166(Arg orHis), Ala172Thr; Ala173Val; Thr174Leu; Ala177Glu; Glu178Asp; Arg179Leu;Val180Gly; Ala201Ser; Val212Leu; Asp213Glu; Val215Ile; Ser226Arg;Lys244Arg; Gln251His; Val254Ile; Ser255Asn; Glu257Gly; Thr264(Ala orIle); Thr272Met; Cys274Gly; Val275Ile; Lys280Arg; Ser281Asn; Lys285Asp;Lys286Arg; Phe290Ser; Thr291(Met or Val); Lys293Gln; Thr297Lys;Ile299(Thr or Val); Arg301His; Asn303Asp; Ser318Phe; Glu321Asp;Pro326Ser; Cys327Leu; and Ser328(Gly or Gln).

In various embodiments, the polypeptide comprises, e.g., at least 15, atleast 20, at least 30, at least 50, at least 75, at least 100, at least150, at least 200, or at least 250 contiguous amino acid residues of anyone of SEQ ID NOS:8-14 or SEQ ID NO:39. In other embodiments, theencoded polypeptide is at least 270, 280, 285, 290, 295, 300, 305, 307,310, or 320 amino acids in length. In another embodiment, the encodedpolypeptide comprises at least 290, 300, 305, or 307 contiguous aminoacid residues of the mature polypeptide region of any one of SEQ IDNOS:8-14 or SEQ ID NO:39.

Fragments of the modified p40 polypeptides described herein are also afeature of the invention. A modified p40 polypeptide fragment of theinvention typically comprises a modified p40 polypeptide comprising atleast about 20, 25, or 30, and typically at least about 40, 50, 60, 70,80, 90, or 100 contiguous amino acids of any one of SEQ ID NOS:8-14 orSEQ ID NO:39. In other embodiments, the fragment comprises usually atleast about 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 290, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, or 307 contiguousamino acids of any one of SEQ ID NOS:8-14 or SEQ ID NO:39. Suchpolypeptide fragments may have proliferative activity in a humanT-cell-based assay and/or interferon-gamma induction activity in a humanT-cell based assay.

In other embodiments, the invention provides polypeptides having alength of at least about 295 amino acids, and in some such embodiments,such polypeptides have a proliferative activity or interferon-gammainduction activity in a T-cell based assay (such as, e.g., a humanT-cell based assay), in the presence of a p35 polypeptide (such as,e.g., a wt-p35 polypeptide or a modified p35 polypeptide).

In other embodiments, the invention provides a polypeptide comprising atleast 280, 290, 295, or 300 contiguous amino acid residues of a proteinencoded by a coding polynucleotide sequence comprising any of thefollowing: (a) SEQ ID NO:1 to SEQ ID NO:7; (b) a coding polynucleotidesequence that encodes a first polypeptide selected from any of SEQ IDNO:8 to SEQ ID NO:14 or SEQ ID NO:39; and (c) a complementarypolynucleotide sequence that hybridizes under at least highly stringent(or ultra-high stringent or ultra-ultra-high stringent conditions)hybridization conditions over substantially the entire length of apolynucleotide sequence of (a) or (b). Such polypeptides may haveproliferative activity or interferon-gamma induction activity in aT-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or amodified p35 polypeptide).

The polypeptides and nucleic acids of the subject invention need not beidentical, but can be substantially identical to the correspondingsequence of the target molecule or related molecule, including thepolypeptides of any of SEQ ID NOS:8-14 or SEQ ID NO:39 or fragmentsthereof (including those having T-cell proliferative or interferon-gammainduction activities in the assays described herein), or the nucleicacids of any of SEQ ID NOS:1-7 or fragments thereof (including thosehaving T-cell proliferative or interferon-gamma induction activities inthe assays described herein). The polypeptides can be subject to variouschanges, such as insertions, deletions, and substitutions, eitherconservative or non-conservative, where such changes might provide forcertain advantages in their use. The polypeptides of the invention canbe modified in a number of ways so long as they comprise a sequencesubstantially identical (as defined below) or having a percent identityto a sequence in the naturally occurring or known p40 polypeptidemolecule.

In other embodiments, the invention provides a modified p40 polypeptidecomprising an amino acid sequence having at least about 90% amino acidsequence identity to the sequence identified herein as the maturepolypeptide region (amino acid residue positions 23-328) of SEQ IDNO:39:IWEL-X₂₇-K-X₂₉-VYVVELDWYP-X₄₀-APGE-X₄₅-VVL-X₄₉-CDTPEEDGITWT-X₆₂-DQSS-X₆₇-VLG-X₇₁-GKTLTI-X₇₈-VKEFGDAGQYTC-X₉₁-KGG-X₉₅-X₉₆-LS-X₉₉-SLLLLHKKEDGIWSTDILKDQK-X₁₂₂-PK-X₁₂₅-K-X₁₂₇-FL-X₁₃₀-CEAK-X₁₃₅-YSG-X₁₃₉-FTCWWLT-X₁₄₇-ISTDL-X₁₅₃-F-X₁₅₅-VKSSRGS-X₁₆₃-DP-X₁₆₆-GVTCG-X₁₇₂-X₁₇₃-X₁₇₄-LS-X₁₇₇-X₁₇₈-X₁₇₉-X₁₈₀-X₁₈₁-X₁₈₂-X₁₈₃-X₁₈₄-X₁₈₅-X₁₈₆-X₁₈₇-X₁₈₈-Y-X₁₉₀-VECQE-X₁₉₆-SACP-X₂₀₁-AEESLPIEV-X₂₁₁-X₂₁₂-X₂₁₃-A-X₂₁₅-HKLKYENYTS-X₂₂₆-FFIRDIIKPDPPKNLQL-X₂₄₄-PLKNSR-X₂₅₁-VE-X₂₅₄-X₂₅₅-W-X₂₅₇-YPDTWS-X₂₆₄-PHSYFSLTF-X₂₇₄-X₂₇₅-QVQG-X₂₈₀-X₂₈₁-KRE-X₂₈₅-X₂₈₆-X₂₈₇-X₂₈₈-X₂₈₉-F-X₂₉₁-D-X₂₉₃-TSA-X₂₉₇-V-X₂₉₉-C-X₃₀₁-K-X₃₀₃-A-X₃₀₅-I-X₃₀₇-V-X₃₀₉-A-X₃₁₁-DRY-X₃₁₅-SS-X₃₁₈-WS-X₃₂₁-WASV-X₃₂₆-X₃₂₇-X₃₂₈,or a conservatively substituted variation thereof, where X₂₇ is K or E;X₂₉ is D or N; X₄₀ is D or N; X₄₅ is M or T; X₄₉ is T or A; X₆₂ is S;X₆₇ is E or G; X₇₁ is T; X₇₈ is H; X₉₁ is H or R; X₉₅ is E, A, K, or T,X₉₆ is V or A; X₉₉ is R or Q; X₁₂₂ is E or K; X₁₂₅ is N or A; X₁₂₇ is Sor I; X₁₃₀ is K; X₁₃₅ is N or D; X₁₃₉ is R or H; X₁₄₇ is T or A; X₁₅₃ isT or K; X₁₅₅ is S or T; X₁₆₃ is S or T; X₁₆₆ is Q, R, or H; X₁₇₂ is A orT; X₁₇₃ is A or V; X₁₇₄ is T or L; X₁₇₇ is A or E; X₁₇₈ is E or D; X₁₇₉is R, L, or K; X₁₈₀ is V or G; X₁₈₁ to X₁₈₄ inclusive is deleted, or isreplaced with the sequence S-(L or M)-(E or D)-H-R; X₁₈₅ is E; X₁₈₆ isY; X₁₈₇ is K or N; X₁₈₈ is K; X₁₉₀ is R or T; X₁₉₆ is G; X₂₀₁ is A or S;X₂₁₁ is V; X₂₁₂ is V or L; X₂₁₃ is D or E; X₂₁₅ is V or I; X₂₂₆ is S orR; X₂₄₄ is K or R; X₂₅₁ is Q or H; X₂₅₄ is V or I; X₂₅₅ is S or N; X₂₅₇is E or G; X₂₆₄ is T or A; X₂₇₄ is C or G; X₂₇₅ is V or I; X₂₈₀ is K orR; X₂₈₁ is S or N; X₂₈₅ is K or D; X₂₈₆ is K or R; X₂₈₇ is D or isdeleted; X₂₈₈ is R or is deleted; X₂₈₉ is I or L; X₂₉₁ is T or M; X₂₉₃is K or Q; X₂₉₇ is T or K; X₂₉₉ is I, T, or V; X₃₀₁ is R or H; X₃₀₃ is Nor D; X₃₀₅ is K; X₃₀₇ is R; X₃₀₉ is Q; X₃₁₁ is R; X₃₁₅ is Y or H; X₃₁₈is S or F; X₃₂₁ is E or D; X₃₂₆ is P or S; X₃₂₇ is C or L; and X₃₂₈ isS, G, or Q. In various embodiments, the modified p40 polypeptidecomprises an amino acid sequence having at least about 90%, 92%, 95%,96%, 97%, 98%, or 99% amino acid sequence identity to the maturepolypeptide region (amino acid residue positions 23-328) of SEQ IDNO:39. In another embodiment, the modified p40 polypeptide comprises anamino acid sequence identified herein as the mature polypeptide region(amino acid residue positions 23-328) of SEQ ID NO:39.

The modified p40 polypeptide may further comprise a leader peptidesequence having at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99%amino acid sequence identity to the amino acid sequenceM-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identified herein as the leaderpeptide region (amino acid residue positions 1-22) of SEQ ID NO:39, or aconservatively substituted variation thereof where X₂ is C or H; X₃ is Hor P; X₈ is I or V; X₁₅ is F or L; and X₂₁ is V or M.

In other embodiments, the present invention provides a modified p40polypeptide comprising a conservatively modified variation of the aminoacid sequence identified herein as the mature polypeptide region (aminoacid residue positions 23-328) of SEQ ID NO:39, and, optionally,comprising a conservatively modified variation of the leader peptideregion (amino acid residue positions 1-22) of SEQ ID NO:39. Theinvention also provides a fragment of said polypeptide havingproliferative activity in a human T-cell based assay or interferon-gammainduction activity in a human T-cell based assay. In other embodiments,the invention includes a modified p40 polypeptide comprising the aminoacid sequence identified herein as the mature polypeptide region (aminoacid residue positions 23-328) of SEQ ID NO:39, and, optionally,comprising the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:39, and a polypeptide or a fragment of said polypeptidehaving proliferative activity or interferon-gamma induction activity ina T-cell based assay (such as, e.g., a human T-cell based assay), in thepresence of a p35 polypeptide (such as, e.g., a wt-p35 polypeptide or amodified p35 polypeptide).

The invention also provides a polypeptide comprising a leader peptidesequence having at least about 90% amino acid sequence identity to theamino acid sequence M-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, identifiedherein as the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:39, where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅ isF or L; and X₂₁ is V or M. In various embodiments, the leader peptidesequence encoded by the nucleic acid of the invention comprises an aminoacid sequence having at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99%amino acid sequence identity to leader peptide region (amino acidresidues 1-22) of SEQ ID NO:39. In other embodiments, the leader peptidesequence comprises the sequence identified as the leader peptide region(amino acid residue positions 1-22) of SEQ ID NO:39, or a conservativelymodified variation thereof. Each of the single letters in the amino acidsequences presented above represents a particular amino acid residue,according to standard practice known to those of ordinary skill in theart.

Modified p35 Polypeptides

An isolated or recombinant modified p35 polypeptide of the inventionincludes a polypeptide comprising a mature polypeptide region of asequence selected from SEQ ID NO:26 to SEQ ID NO:35, conservativelymodified variations thereof, and fragments thereof having proliferativeactivity in a T-cell based assay or interferon-gamma induction activityin a T-cell based assay. An alignment of exemplary modified p35polypeptide sequences according to the invention is provided in FIG. 11.Alignment of the polypeptide sequences of the invention to each other orto sequences of known, naturally-occurring p35 polypeptides is readilyperformed by one of ordinary skill in the art using publicly availabledatabases and alignment programs. Sequences of known,naturally-occurring p35 polypeptides from human and non-human sourcesare readily available from a variety of sources, such as GenBank orSWISSPROT.

The modified p35 polypeptide of the invention comprises at least onemodification at a position equivalent to that in the amino acid sequenceof a human p35 polypeptide (SEQ ID NO:36). The modification can include:

(a) a substitution of the specified amino acid for a different aminoacid at least one equivalent position selected from Thr91, Met120,Ala121, Val212, Thr213, and Ala218;

(b) a insertion of at least one of the amino acid residues Phe-His-Leubetween equivalent positions Leu19 and Ser20;

(c) a deletion of the amino acid at equivalent position Pro36.

The modified p35 polypeptide may optionally include at least two ofmodification (a), (b) or (c). The modified p35 polypeptide sequence maybe a modified sequence of a naturally-occurring p35 polypeptide sequenceof a mammal (e.g., human, a non-human primate, a ruminant, or a rodent),preferably a primate, more preferably human. For purposes of clarity,the numbering of amino acid positions corresponds to that of a human p35polypeptide sequence (SEQ ID NO:36); however, it is understood that suchmodification may be made in an equivalent position of any p35polypeptide sequence.

An “equivalent position”, or a “position equivalent to”, refers to aposition related by sequence homology to a specified position of thecomparison sequence (in this case, SEQ ID NO:36). For example, in FIG.11 the amino acid sequence of a human p35 polypeptide (SEQ ID NO:36) andexemplary modified p35 polypeptide sequences of the invention (SEQ IDNO:26 to SEQ ID NO:35) are aligned to provide the maximum amount ofhomology between the sequences. A comparison of these sequences showsthat there are a number of conserved residues contained in eachsequence. As can be seen, there are also a number of insertions anddeletions in the exemplary modified p35 sequences as compared to thehuman p35 sequence.

Thus, for example, inspection of FIG. 11 shows that the amino acidposition equivalent to amino acid 23 in human p35 (i.e., the “equivalentposition” to amino acid 23), is 26 in the modified p35 polypeptidesequence SEQ ID NO:26 (owing to the insertion of Pro-His-Lys betweenequivalent position 19 and 20 of SEQ ID NO:36).

In one embodiment, a modified p35 polypeptide of the invention comprisesat least one substitution at a position equivalent to that of the aminoacid sequence of human p35 polypeptide (SEQ ID NO:36), selected from thegroup consisting of: Thr91(Ala or Ile), Met120Thr, Ala121Thr, Val212Met,Thr213Met, and Ala218Ser. In other embodiments, a modified p35polypeptide of the invention comprises at least 2, 3, 4, or 5 of theabove substitutions. In another embodiment, a modified p35 polypeptidecomprises all of the above substitutions.

A modified p35 polypeptide of the invention optionally further comprisesat least one of the following substitutions in a position equivalent tothat of SEQ ID NO:36: Cys2Tyr; Ala4(Leu or Pro); Ser6Gly; Val10Ile;Ala11Ser; Asp17H is; Ala22Gly; Asn24Ser; Val27Thr; Ala28Thr; Pro30Ala;Asp31(Ser or Gly); Met34Arg; Phe35(Ser or Leu); Pro36(deleted);His39Asp; His40Tyr; Arg46Lys; Val48Ala; Met51 Thr; Lys54Arg; Thr58Ile;Pro63Ser; Ile69Thr; Lys76Gln; Lys92Thr; Asn98Ala; Glu101Gly; Thr102Ile;Phe104Leu; Leu124His; Ser125Gly; Val136Met; Thr140Ala; Asp148Asn;Ala161Thr; Val162Ala; Asp164Ala; Met167Leu; Phe172Val; Val177Ala;Ser181Pro; Pro186Leu; Asp210Asn; and insertion of one or more of 220Leu;221Glu; 222Ser; and 223Ser.

The modified p35 polypeptide sequence may be a modification of anaturally-occurring p35 polypeptide sequence of a mammal (e.g., human, anon-human primate, a ruminant, or a rodent), preferably a primate, morepreferably human. The naturally-occurring p35 polypeptide sequence maybe one encoded by one of the following nucleic acid sequences havingGenBank accession number: M65271, M65291 (human); U19842 (Macacamulatta, rhesus monkey), U19835 (Cercocebus torquatus, sooty mangabey),U83185, Y07761, AF054605 (Felis catus, cat), U49085 (Canis familiaris,dog), L35765 (Sus scrofa, pig), Y11130 (Equus caballus, horse), U14416(Bos taurus, cow), U57751 (Cervus elaphus, red deer), AF173557 (Ovisaries, sheep), AF003542 (Capra hircus, goat), X97018 (Marmota monax,woodchuck), AF177031 (Rattus norvegicus, rat), and M86672, S82419 (Musmusculus, mouse). The naturally-occurring p35 polypeptide sequence ismore preferably a primate sequence, still more preferably a humansequence, most preferably a mature human p35 sequence comprising aminoacid residues 23 to 219 of SEQ ID NO:36.

The invention also includes an isolated or recombinant polypeptidecomprising an amino acid sequence containing at least 10 contiguousamino acid residues of any one of SEQ ID NOS:26-35. The polypeptidetypically includes one or more amino acid substitution, at an equivalentposition to that of SEQ ID NO:36, selected from: Thr91(Ala or Ile),Met120Thr, Ala121Thr, Val212Met, Thr213Met, and Ala218Ser. Thepolypeptide optionally includes one or more substitution selected from:Cys2Tyr; Ala4(Leu or Pro); Ser6Gly; Val10Ile; Ala11Ser; Asp17His;Ala22Gly; Asn24Ser; Val27Thr; Ala28Thr; Pro30Ala; Asp31(Ser or Gly);Met34Arg; Phe35(Ser or Leu); Pro36(deleted); His39Asp; His40Tyr;Arg46Lys; Val48Ala; Met51Thr; Lys54Arg; Thr58Ile; Pro63Ser; Ile69Thr;Lys76Gln; Lys92Thr; Asn98Ala; Glu101Gly; Thr102Ile; Phe104Leu;Leu124His; Ser125Gly; Val136Met; Thr140Ala; Asp148Asn; Ala161Thr;Val162Ala; Asp164Ala; Met167Leu; Phe172Val; Val177Ala; Ser181Pro;Pro186Leu; Asp210Asn; and insertion of one or more of 220Leu; 221Glu;222Ser; and 223Ser.

In various embodiments, the polypeptide comprises, e.g., at least 15, atleast 20, at least 30, at least 50, at least 75, at least 100, at least150, at least 170, or at least 180 contiguous amino acid residues of anyone of SEQ ID NOS:26-35 and SEQ ID NO:40. In another embodiment, thepolypeptide comprises at least 185, 190, 195, or 200 contiguous aminoacid residues of the mature polypeptide region of any one of SEQ IDNOS:26-35 and SEQ ID NO:40.

Fragments of the modified p35 polypeptides described herein are also afeature of the invention. A modified p35 polypeptide fragment of theinvention typically comprises a modified p35 polypeptide comprising atleast about 20, 25, or 30, and typically at least about 40, 50, 60, 70,80, 90, or 100 contiguous amino acid residues of any one of SEQ IDNOS:26-35 or SEQ ID NO:40. In other embodiments, the fragment comprisesusually at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 185,190, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or205 contiguous amino acid residues of any one of SEQ ID NOS:8-14 or SEQID NO:40. Such polypeptide fragments may have proliferative activity orinterferon-gamma induction activity in a T-cell based assay (such as,e.g., a human T-cell based assay), in the presence of a p40 polypeptide(such as, e.g., a wt-p40 polypeptide or a modified p40 polypeptide).

In other embodiments, the invention provides polypeptides having alength of at least about 190 amino acids, and in some such embodiments,such polypeptides have proliferative activity or interferon-gammainduction activity in a T-cell based assay (such as, e.g., a humanT-cell based assay), in the presence of a p40 polypeptide (such as,e.g., a wt-p40 polypeptide or a modified p40 polypeptide)

In other embodiments, the invention provides a polypeptide comprising atleast 190, 195, or 200 contiguous amino acid residues of a proteinencoded by a coding polynucleotide sequence comprising any of thefollowing: (a) SEQ ID NO:16 to SEQ ID NO:25; (b) a coding polynucleotidesequence that encodes a first polypeptide selected from any of SEQ IDNO:26 to SEQ ID NO:35 or SEQ ID NO:40; and (c) a complementarypolynucleotide sequence that hybridizes under at least highly stringent(or ultra-high stringent or ultra-ultra-high stringent conditions)hybridization conditions over substantially the entire length of apolynucleotide sequence of (a) or (b). Such polypeptides may haveproliferative activity in a T-cell-based assay (or other similar assay),and/or interferon-gamma induction activity in a T-cell-based assay (orother similar assay). The polypeptides and nucleic acids of the subjectinvention need not be identical, but can be substantially identical tothe corresponding sequence of the target molecule or related molecule,including the polypeptides of any of SEQ ID NOS:26-35 or SEQ ID NO:40 orfragments thereof (including those having T-cell proliferative orinterferon-gamma induction activities in the assays described herein),or the nucleic acids of any of SEQ ID NOS:16-25 or fragments thereof(including those having T-cell proliferative or interferon-gammainduction activities in the assays described herein). The polypeptidescan be subject to various changes, such as insertions, deletions, andsubstitutions, either conservative or non-conservative, where suchchanges might provide for certain advantages in their use. Thepolypeptides of the invention can be modified in a number of ways solong as they comprise a sequence substantially identical (as definedbelow) or having a percent identity to a sequence in the naturallyoccurring or known p35 polypeptide molecule.

In other embodiments, the invention provides a modified p35 polypeptidecomprising an amino acid sequence having at least about 90% amino acidsequence identity to the sequence identified herein as the maturepolypeptide region (amino acid residue positions 23-219) of SEQ IDNO:40:R-X₂₄-LP-X₂₇-X₂₈-T-X₃₀-X₃₁-PG-X₃₄-X₃₅-X₃₆-CL-X₃₉-X₄₀-SQNLL-X₄₆-A-X₄₈-SN-X₅₁-LQ-X₅₄-A-X₅₆-Q-X₅₈-LEFY-X₆₃-CTSEE-X₆₉-DHEDIT-X₇₆-DKTSTVEACLPLEL-X₉₁-X₉₂-NESCL-X₉₈-SR-X₁₀₁-X₁₀₂-S-X₁₀₄-ITNGSCLASRKTSFM-X₁₂₀-X₁₂₁-LC-X₁₂₄-X₁₂₅-SIYEDLKMYQ-X₁₃₆-EFK-X₁₄₀-MNAKLLM-X₁₄₈-PKRQIFLDQNML-X₁₆₁-X₁₆₂-I-X₁₆₄-EL-X₁₆₇-QALN-X₁₇₂-NSET-X₁₇₇-PQK-X₁₈₁-SLEE-X₁₈₆-DFYKTKIKLCILLHAFRIRAVTI-X₂₁₀-R-X₂₁₂-X₂₁₃-SYLN-X₂₁₈-S,or a conservatively substituted variation thereof, where X₂₄ is N or S;X₂₇ is V or T; X₂₈ is A or T; X₃₀ is P or A; X₃₁ is D, S, or G; X₃₄ is Mor R; X₃₅ is F, S, or L; X₃₆ is P or is deleted; X₃₉ is H or D; X₄₀ is Hor Y; X₄₆ is R or K; X₄₈ is V or A; X₅₁ is M or T; X₅₄ is K or R; X₅₆ isK or R; X₅₈ is T or I; X₆₃ is P or S; X₆₉ is I or T; X₇₆ is K or Q; X₉₁is A or I; X₉₂ is K or T; X₉₈ is N or A; X₁₀₁ is E or G; X₁₀₂ is T or I;X₁₀₄ is F or L; X₁₂₀ is T; X₁₂₁ is T; X₁₂₄ is L or H; X₁₂₅ is S or G;X₁₃₆ is V or M; X₁₄₀ is T or A; X₁₄₈ is D or N; X₁₆₁ is A or T; X₁₆₂ isV or A; X₁₆₄ is D or A; X₁₆₇ is M or L; X₁₇₂ is For V; X₁₇₇ is V or A;X₁₈₁ is S or P; X₁₈₆ is P or L; X₂₁₀ is D or N; X₂₁₂ is M; X₂₁₃ is M;and X₂₁₈ is S. In various embodiments, the modified p35 polypeptidecomprises an amino acid sequence having at least about 90%, 92%, %, 95%,96%, 97%, 98%, or 99% amino acid sequence identity to the maturepolypeptide region (amino acid residue positions 23-219) of SEQ IDNO:40. The invention also includes a polynucleotide sequence encodingsaid polypeptide or a fragment of said polypeptide having proliferativeactivity in a human T-cell based assay or interferon-gamma inductionactivity in a human T-cell based assay.

The modified p35 polypeptide of the invention may further comprise aleader peptide sequence having at least about 90%, 92%, 95%, 96%, 97%,98%, or 99% amino acid sequence identity to the amino acid sequenceM-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₇-HLSL-X₂₂, identified herein as theleader peptide region (amino acid residue positions 1-22) of SEQ IDNO:40, or a conservatively substituted variation thereof, where X₂ is Cor Y; X₄ is A, L or P; X₆ is S or G; X₁₀ is V or I; X₁₁ is A or S; X₁₇is D or H; and X₂₂ is A or G, and optionally includes an insertion ofthe amino acids P-H-L between positions 18 and 19. Each of the singleletters in the amino acid sequences presented above represents aparticular amino acid residue, according to standard practice known tothose of ordinary skill in the art.

The present invention also includes a modified p35 polypeptidecomprising a conservatively modified variation of the amino acidsequence identified herein as the mature polypeptide region (amino acidresidue positions 23-219) of SEQ ID NO:40, and, optionally, aconservatively modified variation of the leader peptide region (aminoacid residue positions 1-22) of SEQ ID NO:40, and a polynucleotidesequence encoding said polypeptide or a fragment of said polypeptidehaving proliferative activity in a human T-cell based assay orinterferon-gamma induction activity in a human T-cell based assay. Theinvention also includes a modified p35 polypeptide comprising the aminoacid sequence identified herein as the mature polypeptide region (aminoacid residue positions 23-219) of SEQ ID NO:40, and, optionally,comprising the leader peptide region (amino acid residue positions 1-22)of SEQ ID NO:40, and a polypeptide or a fragment of said polypeptidehaving proliferative activity in a human T-cell based assay orinterferon-gamma induction activity in a human T-cell based assay.

The invention also includes a polypeptide comprising a leader peptidesequence having at least about 90% amino acid sequence identity to theamino acid sequence M-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₆-HLSL-X₂₂,identified herein as the leader peptide region (amino acid residuepositions 1-22) of SEQ ID NO:40, where X₂ is C or Y; X₄ is A, L, or P;X₆ is S or G; X₁₀ is V or I; X₁₁ is A or S; X₁₆ is D or H; X₂₂ is A orG, and optionally includes an insertion of the amino acids P-H-L betweenpositions 18 and 19. In various embodiments, the leader peptide sequencecomprises an amino acid sequence having at least about 90%, 92%, 95%,96%, 97%, 98%, or 99% amino acid sequence identity to leader peptideregion (amino acid residue positions 1-22) of SEQ ID NO:40. In anotherembodiment, the leader peptide sequence comprises the sequenceidentified as the leader peptide region (amino acid residue positions1-22) of SEQ ID NO:40, or a conservatively modified variation thereof.Each of the single letters in the amino acid sequences presented aboverepresents a particular amino acid residue, according to standardpractice known to those of ordinary skill in the art.

Sequence Variations

The polypeptides and nucleic acids of the subject invention need not beidentical, but can be substantially identical to the correspondingsequence of the target molecule or related molecule, including thepolypeptides of any of SEQ ID NOS:8-14, 26-35, 37, 38 or fragmentsthereof (including those having T-cell proliferative or interferon-gammainduction activities in the assays described herein), or the nucleicacids of any of SEQ ID NOS:1-7, SEQ ID NOS:16-25 or fragments thereof(including those having T-cell proliferative or interferon-gammainduction activities in the assays described herein). The polypeptidescan be subject to various changes, such as insertions, deletions, andsubstitutions, either conservative or non-conservative, where suchchanges might provide for certain advantages in their use. Thepolypeptides of the invention can be modified in a number of ways solong as they comprise a sequence substantially identical (as definedbelow) or having a percent identity to a sequence in the naturallyoccurring or known p40 or p35 polypeptide molecule.

Alignment and comparison of relatively short amino acid sequences (lessthan about 30 residues) is typically straightforward. Comparison oflonger sequences can require more sophisticated methods to achieveoptimal alignment of two sequences. Optimal alignment of sequences foraligning a comparison window can be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman(1988) Proc. Nat'l Acad. Sci. (USA) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of sequencesimilarity over the comparison window) generated by the various methodsis selected.

The term sequence identity means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over a window ofcomparison. The term “percentage of sequence identity” or “percentsequence identity” is calculated by comparing two optimally alignedsequences over the window of comparison, determining the number ofpositions at which the identical residues occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. In one aspect, the present inventionprovides modified p40 nucleic acids having at least about 90%, 92%, 95%,96%, 97%, 98%, 99%, 99.5% or more percent sequence identity with thenucleic acids of any of SEQ ID NOS:1-7 or fragments thereof. In anotheraspect, the present invention provides modified p35 nucleic acids havingat least about 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5% or more percentsequence identity with the nucleic acids of any of SEQ ID NOS:16-25 orfragments thereof.

As applied to polypeptides, the term substantial identity means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights (described in detail below), shareat least about 80 percent sequence identity, preferably at least about85 percent sequence identity, more preferably at least about 90 percentsequence identity or more (e.g., 92, 95, 96, 97, 98, or 99 percentsequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions typically refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Some preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. In one aspect, the present invention providesmodified p40 polypeptides having at least about 90%, 92%, 95%, 96%, 97%,98% 99% 99.5% or more percent sequence identity with the polypeptides ofany of SEQ ID NOS:8-14, SEQ ID NO:39, or fragments thereof. In anotheraspect, the present invention provides modified p35 polypeptides havingat least about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% 99% 99.5% or morepercent sequence identity with the polypeptides of any of SEQ IDNOS:26-35, SEQ ID NO:40, or fragments thereof.

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the FASTAalgorithm, which is described in Pearson, W. R. & Lipman, D. J., 1988,Proc. Nat'l Acad. Sci. USA 85: 2444. See also W. R. Pearson, 1996,Methods Enzymol. 266: 227-258. Preferred parameters used in a FASTAalignment of DNA sequences to calculate percent identity are optimized,BL50 Matrix 15: −5, k-tuple=2; joining penalty=40, optimization=28; gappenalty −12, gap length penalty=−2; and width=16.

Another preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc.Acids Res. 25: 3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used with the parametersdescribed herein to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Nat'l Acad. Sci. U.S.A. 89: 10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul (1993)Proc. Nat'l Acad. Sci. U.S.A. 90: 5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle(1987) J. Mol. Evol. 35: 351-360. The method used is similar to themethod described by Higgins & Sharp (1989) CABIOS 5: 151-153. Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal. (1984) Nuc. Acids Res. 12: 387-395.

Another preferred example of an algorithm that is suitable for multipleDNA and amino acid sequence alignments is the CLUSTALW program(Thompson, J. D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680).CLUSTALW performs multiple pairwise comparisons between groups ofsequences and assembles them into a multiple alignment based onhomology. For the initial pairwise alignments, Gap open and Gapextension penalties were 10 and 0.1, respectively. For the multiplealignments, Gap open penalty was 10, and the Gap extension penalty was0.05. For amino acid alignments, the BLOSUM62 substitution matrix can beused as a protein weight matrix (Henikoff and Henikoff (1992) Proc.Nat'l Acad. Sci. U.S.A. 89: 10915-10919). CLUSTALW can be obtained from,for example, the Vector NTI sequence analysis suite, e.g., version 6(InforMax, Inc., North Bethesda, Md.).

Conservatively Modified Variations

Polypeptides of the present invention include conservatively modifiedvariations of the sequences disclosed herein as SEQ ID NO:8 to SEQ IDNO:14 and SEQ ID NO:26 to SEQ ID NO:35. Such conservatively modifiedvariations comprise substitutions, additions or deletions which alter,add or delete a single amino acid or a small percentage of amino acids(typically less than about 5%, more typically less than about 4%, 2%, or1%) in any of the mature polypeptide sequences of SEQ ID NO:8 to SEQ IDNO:14 and SEQ ID NO:26 to SEQ ID NO:35.

For example, a conservatively modified variation (e.g., deletion) of the302 amino acid mature polypeptide identified herein as amino acidresidues 23 to 324 SEQ ID NO:8 will have a length of at least 287 aminoacids, preferably at least 290 amino acids, more preferably at least 296amino acids, and still more preferably at least 299 amino acids,corresponding to a deletion of less than about 5%, 4%, 2% or 1% of thepolypeptide sequence.

Another example of a conservatively modified variation (e.g., a“conservatively substituted variation”) of the mature portion of thepolypeptide identified herein as SEQ ID NO: 8 will contain “conservativesubstitutions,” as shown, for example, shown in the six substitutiongroups set forth in Table 5 (supra), in up to about 15 residues (i.e.,less than about 5%) of the 302 amino acid mature polypeptide.

The polypeptides of the invention, including conservatively substitutedsequences, can be present as mature polypeptides (e.g., lacking leadersequences corresponding to amino acid residues 1-22 in each of SEQ IDNO:8 to SEQ ID NO:14 and SEQ ID NO:28 to SEQ ID NO:35, and amino acidresidues 1-25 of SEQ ID NO:26 and SEQ ID NO:27); as comprising any oneof the unique leader sequences identified as amino acid residues 1-22 ofSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:28 andSEQ ID NO:29, and as amino acid residues 1-25 of SEQ ID NO:26 and SEQ IDNO:27); as part of larger polypeptide sequences such as occur upon theaddition of one or more domains for purification of the protein (e.g.,poly-His segments, FLAG epitope segments, etc.), e.g., where theadditional functional domains have little or no effect on the activityof the p35 or p40 portion of the protein, or where the additionaldomains can be removed by post synthesis processing steps such as bytreatment with a protease.

In another embodiment, a mature modified p40 polypeptide of the presentinvention comprises the following sequence:IWEL-X₂₇-K-X₂₉-VYVVELDWYP-X₄₀-APGE-X₄₅-VVL-X₄₉-CDTPEEDGITWT-X₆₂-DQSS-X₆₇-VLG-X₇₁-GKTLTI-X₇₈-VKEFGDAGQYTC-X₉₁-KGG-X₉₅-X₉₆-LS-X₉₉-SLLLLHKKEDGIWSTDILKDQK-X₁₂₂-PK-X₁₂₅-K-X₁₂₇-FL-X₁₃₀-CEAK-X₁₃₅-YSG-X₁₃₉-FTCWWLT-X₁₄₇-ISTDL-X₁₅₃-F-X₁₅₅-VKSSRGS-X₁₆₃-DP-X₁₆₆-GVTCG-X₁₇₂-X₁₇₃-X₁₇₄-LS-X₁₇₇-X₁₇₈-X₁₇₉-X₁₈₀-X₁₈₁-X₁₈₂-X₁₈₃-X₁₈₄-X₁₈₅-X₁₈₆-X_(187-X)₁₈₈-Y-X₁₉₀-VECQE-X₁₉₆-SACP-X₂₀₁-AEESLPIEV-X₂₁₁-X₂₁₂-X₂₁₃-A-X₂₁₅-HKLKYENYTS-X₂₂₆-FFIRIIKPDPPKNLQL-X₂₄₄-PLKNSR-X₂₅₁-VE-X₂₅₄-X₂₅₅-W-X₂₅₇-YPDTWS-X₂₆₄-PHSYFSLTF-X₂₇₄-X₂₇₅-QVQG-X₂₈₀-X₂₈₁-KRE-X₂₈₅-X₂₈₆-X₂₈₇-X₂₈₈-X₂₈₉-F-X₂₉₁-D-X₂₉₃-TSA-X₂₉₇-V-X₂₉₉-C-X₃₀₁-K-X₃₀₃-A-X₃₀₅-I-X₃₀₇-V-X₃₀₉-A-X₃₁₁-DRY-X₃₁₅-SS-X₃₁₈-WS-X₃₂₁-WASV-X₃₂₆-X₃₂₇-X₃₂₈,or a conservatively substituted variation thereof,

where X₂₇ is K or E; X₂₉ is D or N; X₄₀ is D or N; X₄₅ is M or T; X₄₉ isT or A; X₆₂ is S; X₆₇ is E or G; X₇₁ is T; X₇₈ is H; X₉₁ is H or R; X₉₅is E, A, K, or T, X₉₆ is V or A; X₉₉ is R or Q; X₁₂₂ is E or K; X₁₂₅ isN or A; X₁₂₇ is S or I; X₁₃₀ is K; X₁₃₅ is N or D; X₁₃₉ is R or H; X₁₄₇is T or A; X₁₅₃ is T or K; X₁₅₅ is S or T; X₁₆₃ is S or T; X₁₆₆ is Q, R,or H; X₁₇₂ is A or T; X₁₇₃ is A or V; X₁₇₄ is T or L; X₁₇₇ is A or E;X₁₇₈ is E or D; X₁₇₉ is R, L, or K; X₁₈₀ is V or G; X₁₈₁ to X₁₈₄inclusive is deleted or is replaced with the sequence S-(L or M)-(E orD)-H-R; X₁₈₅ is E; X₁₈₆ is Y; X₁₈₇ is K or N; X₁₈₈ is K; X₁₉₀ is R or T;X₁₉₆ is G; X₂₀₁ is A or S; X₂₁₁ is V; X₂₁₂ is V or L; X₂₁₃ is D or E;X₂₁₅ is V or I; X₂₂₆ is S or R; X₂₄₄ is K or R; X₂₅₁ is Q or H; X₂₅₄ isV or I; X₂₅₅ is S or N; X₂₅₇ is E or G; X₂₆₄ is T or A; X₂₇₄ is C or G;X₂₇₅ is V or I; X₂₈₀ is K or R; X₂₈₁ is S or N; X₂₈₅ is K or D; X₂₈₆ is.K or R; X₂₈₇ is D or is deleted; X₂₈₈ is R or is deleted; X₂₈₉ is I orL; X₂₉₁ is T or M; X₂₉₃ is K or Q; X₂₉₇ is T or K; X₂₉₉ is I, T, or V;X₃₀₁ is R or H; X₃₀₃ is N or D; X₃₀₅ is K; X₃₀₇ is R; X₃₀₉ is Q; X₃₁₁ isR; X₃₁₅ is Y or H; X₃₁₈ is S or F; X₃₂₁ is E or D; X₃₂₆ is P or S; X₃₂₇is C or L; and X₃₂₈ is S, G, or Q. As defined above, a conservativelymodified variation of the above sequence can include up to a total ofabout 15 amino acid deletions, insertions, or conservative substitutionsin the 306 amino acid sequence, excluding the positions designated X,which correspond to the amino acid explicitly defined. The polypeptidemay further comprise an N-terminal leader sequenceM-X₂-X₃-QQLV-X₈-SWFSLV-X₁₅-LASPL-X₂₁-A, or a conservatively modifiedvariation thereof, where X₂ is C or H; X₃ is H or P; X₈ is I or V; X₁₅is F or L; and X₂₁ is V or M.

In another embodiment, a mature modified p35 polypeptide of the presentinvention comprises the following sequence:R-X₂₄-LP-X₂₇-X₂₈-T-X₃₀-X₃₁-PG-X₃₄-X₃₅-X₃₆-CL-X₃₉-X₄₀-SQNLL-X₄₆-A-X₄₈-SN-X₅₁-LQ-X₅₄-A-X₅₆-Q-X₅₈-LEFY-X₆₃-CTSEE-X₆₉-DHEDIT-X₇₆-DKTSTVEACLPLEL-X₉₁-X₉₂-NESCL-X₉₈-SR-X₁₀₁-X₁₀₂-S-X₁₀₄-ITNGSCLASRKTSFM-X₁₂₀-X₁₂₁-LC-X₁₂₄-X₁₂₅-SIYEDLKMYQ-X₁₃₆-EFK-X₁₄₀-MNAKLLM-X₁₄₈-PKRQIFLDQNML-X₁₆₁-X₁₆₂-I-X₁₆₄-EL-X₁₆₇-QALN-X₁₇₂-NSET-X₁₇₇-PQK-X₁₈₁-SLEE-X₁₈₆-DFYKTKIKLCILLHAFRIRAVTI-X₂₁₀-R-X₂₁₂-X₂₁₃-SYLN-X₂₁₈-S,or a conservatively substituted variation thereof,

where X₂₄ is Nor S; X₂₇ is V or T; X₂₈ is A or T; X₃₀ is P or A; X₃₁ isD, S, or G; X₃₄ is M or R; X₃₅ is F, S, or L; X₃₆ is P or is deleted;X₃₉ is H or D; X₄₀ is H or Y; X₄₆ is R or K; X₄₈ is V or A; X₅₁ is M orT; X₅₄ is K or R; X₅₆ is K or R; X₅₈ is T or I; X₆₃ is P or S; X₆₉ is Ior T; X₇₆ is K or Q; X₉₁ is A or I; X₉₂ is K or T; X₉₈ is N or A; X₁₀₁is E or G; X₁₀₂ is T or I; X₁₀₄ is F or L; X₁₂₀ is T; X₁₂₁ is T; X₁₂₄ isL or H; X₁₂₅ is S or G; X₁₃₆ is V or M; X₁₄₀ is T or A; X₁₄₈ is D or N;X₁₆₁ is A or T; X₁₆₂ is V or A; X₁₆₄ is D or A; X₁₆₇ is M or L; X₁₇₂ isF or V; X₁₇₇ is V or A; X₁₈₁ is S or P; X₁₈₆ is P or L; X₂₁₀ is D or N;X₂₁₂ is M; X₂₁₃ is M; and X₂₁₈ is S. As defined above, a conservativelymodified variation of the above sequence can include up to a total ofabout 10 amino acid deletions, insertions, or conservative substitutionsin the 219 amino acid sequence, excluding the positions designated X,which correspond to the amino acid explicitly defined. The polypeptidemay further comprise the N-terminal leader sequenceM-X₂-P-X₄-R-X₆-LLL-X₁₀-X₁₁-TLVLL-X₁₇-HLSL-X₂₂, or a conservativelymodified variation thereof, where X₂ is C or Y; X₄ is A, L or P; X₆ is Sor G; X₁₀ is V or I; X₁₁ is A or S; X₁₇ is D or H; and X₂₂ is A or G.

Making Polypeptides of the Invention

Recombinant methods for producing and isolating polypeptides of theinvention are described above. In addition to recombinant production,the polypeptides may be produced by direct peptide synthesis usingsolid-phase techniques (cf Stewart et al. (1969) Solid-Phase PeptideSynthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J. Am.Chem. Soc. 85:2149-2154). Peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to providefull-length polypeptides. Fragments of the polypeptides of theinvention, as discussed in greater detail above, are also a feature ofthe invention and may be synthesized by using the procedures describedabove.

Polypeptides of the invention can be produced by introducing into apopulation of cells a nucleic acid of the invention, wherein the nucleicacid is operatively linked to a regulatory sequence effective to producethe encoded polypeptide, culturing the cells in a culture medium toproduce the polypeptide, and optionally isolating the polypeptide fromthe cells or from the culture medium.

In another aspect, polypeptides of the invention can be produced byintroducing into a population of cells a recombinant expression vectorcomprising at least one nucleic acid of the invention, wherein the atleast one nucleic acid is operatively linked to a regulatory sequenceeffective to produce the encoded polypeptide, culturing the cells in aculture medium under suitable conditions to produce the polypeptideencoded by the expression vector, and optionally isolating thepolypeptide from the cells or from the culture medium.

Using Polypeptides of the Invention

Antibodies

In another aspect of the invention, a polypeptide of the invention isused to produce antibodies which have, e.g., diagnostic and therapeuticuses, e.g., related to the activity, distribution, and expression of p35sequences or p40 sequences.

Antibodies to polypeptides of the invention may be generated by methodswell known in the art. Such antibodies may include, but are not limitedto, polyclonal, monoclonal, chimeric, humanized, single chain, Fabfragments and fragments produced by an Fab expression library.Antibodies, i.e., those which block receptor binding, are especiallypreferred for therapeutic use.

Polypeptides for antibody induction do not require biological activity;however, the polypeptide or oligopeptide must be antigenic. Peptidesused to induce specific antibodies may have an amino acid sequenceconsisting of at least 10 amino acids, preferably at least 15 or 20amino acids. Short stretches of a polypeptide of the invention may befused with another protein, such as keyhole limpet hemocyanin, andantibody produced against the chimeric molecule.

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art, and many antibodies are available. See, e.g.,Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497. Other suitable techniques forantibody preparation include selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specificmonoclonal and polyclonal antibodies and antisera will usually bind witha K_(D) of at least about 0.1 μM, preferably at least about 0.01 μM orbetter, and most typically and preferably, 0.001 μM or better.

Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed) (1995) Antibody Engineering, 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,N.J. (Paul).

In one useful embodiment, this invention provides for fully humanizedantibodies against the polypeptides of the invention. Humanizedantibodies are especially desirable in applications where the antibodiesare used as therapeutics in vivo in human patients. Human antibodiesconsist of characteristically human immunoglobulin sequences. The humanantibodies of this invention can be produced in using a wide variety ofmethods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, andBorrebaeck McCafferty and Paul, supra, for a review). In one embodiment,the human antibodies of the present invention are produced initially intrioma cells. Genes encoding the antibodies are then cloned andexpressed in other cells, such as nonhuman mammalian cells. The generalapproach for producing human antibodies by trioma technology isdescribed by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. Theantibody-producing cell lines obtained by this method are called triomasbecause they are descended from three cells; two human and one mouse.Triomas have been found to produce antibody more stably than ordinaryhybridomas made from human cells.

Adjuvants

In one aspect, the modified p40 polypeptides and/or the modified p35polypeptides of the present invention or fragments thereof are useful asadjuvants to stimulate, enhance, potentiate, or augment an immuneresponse related to an antigen when administered together with theantigen or after or before delivery of the antigen. In another aspect,the invention provides methods for administering one or more of thepolypeptides invention described herein to a subject.

Therapeutic and Prophylactic Agents

As described in greater detail herein, the modified p40 polypeptidesand/or the modified p35 polypeptides of the present invention orfragments thereof are useful in the prophylactic and/or therapeutictreatment of a variety of diseases, disorders, or medical conditions.

For example, the invention provides modified p40 polypeptides and/ormodified p35 polypeptides (and nucleic acids which encode suchpolypeptides) that have both T-cell proliferation and interferon-gammainduction activities in the assays described herein.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences as compared to other p35 and p40 sequences, thepolypeptides also provide a new structural features which can berecognized, e.g., in immunological assays. The generation of antiserawhich specifically binds the polypeptides of the invention, as well asthe polypeptides which are bound by such antisera, are a feature of theinvention. The invention includes polypeptides that specifically bind toor that are specifically immunoreactive with an antibody or antiseragenerated against an immunogen comprising an amino acid sequenceselected from one or more of SEQ ID NO:8 to SEQ ID NO:14 and SEQ IDNO:26 to SEQ ID NO:35. To eliminate cross-reactivity with other p40polypeptides, the antibody or antisera is subtracted with availableknown p40 polypeptides, such as those p40 polypeptides encoded bynucleic acids represented by GenBank accession numbers: M65272 andM65290 (human), U19841 (Macaca mulatta, rhesus monkey), U19834(Cercocebus torquatus, sooty mangabey), Y11129 (Equus caballus, horse),U83184, Y07762 and AF054607 (Felis catus, cat), U49100 and AF091134(Canis familiaris, dog), U57752 and U10160 (Cervus elaphus, red deer),AF007576 (Capra hircus, goat), AF004024 (Ovis aries, sheep), U11815 (Bostaurus, cow), U08317 (Sus scrofa, pig), X97019 and AF082494 (Marmotamonax, woodchuck), AF133197 and U16674 (Rattus norvegicus, rat), M86671and S82426 (Mus musculus, mouse), AF097507 (Cavia porcellus, guineapig), and AF046211 (Mesocricetus auratus, golden hamster) or any otherknown p40 polypeptides. Likewise, to eliminate cross-reactivity withother p35 polypeptides, the antibody or antisera is subtracted withavailable known p35 polypeptides, such as those p35 polypeptides encodedby nucleic acids represented by GenBank accession numbers M65271, M65291(human); U19842 (Macaca mulatta, rhesus monkey), U19835 (Cercocebustorquatus, sooty mangabey), U83185, Y07761, AF054605 (Felis catus, cat),U49085 (Canis familiaris, dog), L35765 (Sus scrofa, pig), Y11130 (Equuscaballus, horse), U14416 (Bos taurus, cow), U57751 (Cervus elaphus, reddeer), AF173557 (Ovis aries, sheep), AF003542 (Capra hircus, goat),X97018 (Marmota monax, woodchuck), AF177031 (Rattus norvegicus, rat),and M86672, S82419 (Mus musculus, mouse), or any other known p35polypeptides. These sequences are referred to herein as “the control(p40 or p35) polypeptides”. Where the accession number corresponds to anucleic acid, a polypeptide encoded by the nucleic acid is generated andused for antibody/antisera subtraction purposes. Where the nucleic acidcorresponds to a non-coding sequence, e.g., a pseudo gene, an amino acidwhich corresponds to the reading frame of the nucleic acid is generated(e.g., synthetically), or is minimally modified to include a start codonfor recombinant production.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising sequencescorresponding to one or more of: SEQ ID NO:8 to SEQ ID NO:14 and SEQ IDNO:26 to SEQ ID NO:35, or a substantial subsequence thereof (i.e., atleast about 30%, 40%, 50%, 60%, 70%, 80%; 90%, 95% or 98% of the fulllength sequence provided). The full set of potential polypeptideimmunogens derived from SEQ ID NO:8 to SEQ ID NO:14 and SEQ ID NO:26 toSEQ ID NO:35 are collectively referred to below as “the immunogenicpolypeptides.” The resulting antisera is optionally selected to have lowcross-reactivity against the control p40 polypeptides or the control p35polypeptides, and/or other known p40 or p35 polypeptides, and any suchcross-reactivity is removed by immunoabsorbtion with one or more of thecontrol p40 or p35 polypeptides and/or other known p40 or p35polypeptides, prior to use of the polyclonal antiserum in theimmunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein may be produced in a mammaliancell line. An inbred strain of mice (used in this assay because resultsare more reproducible due to the virtual genetic identity of the mice)is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity).Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide(s) in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control polypeptides to producesubtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control polypeptides. Preferably at least two ofthe immunogenic polypeptides are used in this determination, preferablyin conjunction with at least two of the control polypeptides, toidentify antibodies which are specifically bound by the immunogenicpolypeptide(s).

In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic polypeptides ascompared to binding to the control polypeptides. That is, the stringencyof the binding reaction is adjusted by the addition of non-specificcompetitors such as albumin or non-fat dry milk, or by adjusting saltconditions, temperature, or the like. These binding conditions are usedin subsequent assays for determining whether a test polypeptide isspecifically bound by the pooled subtracted polyclonal antisera. Inparticular, test polypeptides which show at least a 2-5× higher signalto noise ratio than the control polypeptides under discriminatorybinding conditions, and at least about a ½ signal to noise ratio ascompared to the immunogenic polypeptide(s), shares substantialstructural similarity with the immunogenic polypeptide as compared toknown p40 or p35 polypeptides, and is, therefore a polypeptide of theinvention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized polypeptide(s) is compared tothe ability of the immunogenic polypeptide(s) added to the assay tocompete for binding (the immunogenic polypeptides compete effectivelywith the immobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is determined as compared tothe ability of the immunogenic polypeptide(s) to compete for binding tothe antisera. Again, the percent cross-reactivity for the controlpolypeptides is calculated, using standard calculations. Where thepercent cross-reactivity is at least 5-10× as high for the testpolypeptides, the test polypeptides are said to specifically bind thepooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic polypeptide(s). In order to make thiscomparison, the two polypeptides are each assayed at a wide range ofconcentrations and the amount of each polypeptide required to inhibit50% of the binding of the subtracted antisera to the immobilizedpolypeptide is determined using standard techniques. If the amount ofthe test polypeptide required is less than twice the amount of theimmunogenic polypeptide that is required, then the test polypeptide issaid to specifically bind to an antibody generated to the immunogenicpolypeptide, provided the amount is at least about 5-10× as high as fora control polypeptide.

As a final determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorption is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic polypeptide.

Properties of Modified p40 Polypeptides and Modified p35 Polypeptides ofthe Invention

Any polypeptide of the invention may optionally form a dimer (e.g., aheterodimer or a homodimer) with either a p35 or a p40 polypeptide. Asused herein, a “heterodimer” comprises a p35 and a p40 polypeptide, atleast one of which may be a modified p35 polypeptide or a modified p40polypeptide of the invention, while a “homodimer” comprises either twop40 polypeptides or two p35 polypeptides, at least one of which may be amodified p40 polypeptide or a modified p35 polypeptide of the invention.A homodimer need not comprise two identical polypeptides; for example, ap40 homodimer may comprise a naturally-occurring or wild-type p40polypeptide and a modified p40 polypeptide, or two different modifiedp40 polypeptides. A heterodimer comprising a p35 polypeptide and a p40polypeptide typically has an apparent molecular weight of ˜70 to 75 kDa(see, for example, FIGS. 7 and 8); however, the apparent molecularweight may be higher or lower, depending on many factors, such as, forexample, the length of the polypeptides forming the dimer, or the extentof glycosylation of the polypeptides.

A p35 polypeptide which forms a dimer (i.e., “dimerizes”) with apolypeptide of the invention may be a naturally-occurring or wild-typep35 polypeptide, such as a human p35 polypeptide having a sequencecomprising the mature polypeptide region of SEQ ID NO:36, or may be amodified p35 polypeptide, such as a modified p35 polypeptide of thepresent invention, such as, for example, a modified p35 polypeptidehaving a sequence comprising the mature polypeptide region of one of SEQID NO:26 to SEQ ID NO:35 and SEQ ID NO:40. Likewise, a p40 polypeptidewhich forms a dimer (i.e., “dimerizes”) with a polypeptide of theinvention may be a naturally-occurring or wild-type p40 polypeptide,such as a human p40 polypeptide having a sequence comprising the maturepolypeptide region of SEQ ID NO:16, or may be a modified p40polypeptide, such as a modified p40 polypeptide of the presentinvention, such as, for example, a modified p40 polypeptide having asequence comprising the mature polypeptide region of one of SEQ ID NO:8to SEQ ID NO:15 and SEQ ID NO:39.

A composition comprising a polypeptide of the invention (optionally incombination with a corresponding partner subunit polypeptide, e.g., ap35 polypeptide or p40 polypeptide as described above) may optionallyexhibit one or more of the following activities: (a) T-cellproliferative activity, (b) IFN-γ induction activity, (c) Natural Killer(NK) cell-mediated toxicity enhancement activity.

A polypeptide of the invention has “T-cell proliferative activity” whenthe polypeptide or a composition comprising the polypeptide inducesproliferation of phytohemagglutinin-activated T-lymphocytes, asevidenced by, for example, an increase in the ³H-thymidine incorporationof activated T-cells incubated in the presence of ³H-thymidine and thepolypeptide or composition thereof, compared to activated T-cellsincubated with ³H-thymidine alone.

A polypeptide of the invention has “IFN-γ induction activity” when thepolypeptide or a composition comprising the polypeptide induces IFN-γsynthesis in T-lymphocytes (i.e., T-cells), as evidenced by, forexample, an increase in IFN-γ in culture media of T-cells incubated inthe presence of the polypeptide or composition thereof, compared toactivated T-cells incubated without the polypeptide or compositionthereof, respectively. IFN-γ in the culture medium may be assayed by anystandard method, such as by using a commercially-available IFN-γ ELISAkit. Interferon-gamma induction activity in T-cells is indicative ofdifferentiation of T-cells to the T_(H)1 phenotype.

A polypeptide of the invention has “NK cell toxicity enhancementactivity” when the polypeptide or a composition comprising thepolypeptide enhances NK cell-mediated toxicity against a target cell.Enhancement of NK cell toxicity is evidenced by, for example, anincrease in ⁵¹Cr release into the culture supernatant from ⁵¹Cr-labeledtarget cells incubated with T-lymphocytes previously treated with thepolypeptide or composition thereof, compared to that of ⁵¹Cr-labeledtarget cells incubated with T-lymphocytes which were not previouslytreated with the polypeptide or composition thereof, respectively.

The biological activities of exemplary polypeptides of the inventionwere examined as described in the Examples. The results indicate that acomposition comprising a polypeptide of the invention may be used, forexample, to induce proliferation of T-cells, to induce differentiationof naive T cells to T_(H)1 cells, to induce production of IFN-γ inlymphocytes, in in vitro, ex vivo or in vivo applications. Cytokinesthat induce IFN-γ production in vitro typically also enhancecytotoxicity of NK cells.

A polypeptide or nucleic acid of the invention or a compositioncomprising a polypeptide or a nucleic acid of the invention may be usedin methods to promote cell-mediated immunity to a variety of infectiousagents, such as bacterial, protozoal, intracellular parasitic, and viralinfections, particularly in individuals who are highly susceptible tosuch infections, including patients undergoing surgery, patients withtransiently or chronically impaired immune systems due to disease ordrug therapy (e.g., chemotherapy, radiation or immunosuppressivetreatments), individuals infected with HIV or otherwise manifestsymptoms of AIDS or ARC, as well as elderly or otherwiseimmuno-compromised individuals who have reduced capacity to defendagainst opportunistic or infectious microorganisms. In particular, acomposition comprising a polypeptide or a nucleic acid of the inventionmay be useful as a vaccine adjuvant, to enhance a vaccinated host'scell-mediated immunity for a protective response to a pathogen.

Malignant diseases are another useful target for treatment with modifiedp40/p35 heterodimers. Because IFN-γ activates NK cells and cytotoxic Tcells, and induces cytokine production by these cells, the modifiedp40/p35 heterodimers are expected to have potent anti-tumor activitiesin vivo. Cutaneous T cell lymphoma and head and neck cancer may beparticularly useful targets for the modified p40/p35 heterodimertherapies because of low p40/p35 heterodimer production by patients withcutaneous T cell lymphoma and because of good accessibility of thesetumors.

Because IFN-γ enhances T_(H)1 cell differentiation (Chatelain et al.(1992) J. Immunol. 148:1182-1187; De Vries and Punnonen (1996) InCytokine regulation of hu moral immunity: basic and clinical aspects.Eds. Snapper, C. M., John Wiley & Sons, Ltd., West Sussex, UK, p.195-215), and because the modified p40/p35 heterodimers enhanced IFN-γproduction, allergies and asthma are very promising targets for modifiedp40/p35 heterodimer therapies.

Individuals with mutations in their genes encoding natural p40 subunithave dramatically impaired capacity to produce p40, and they aresusceptible to infectious with intracellular pathogens. Treatments witha modified p40/p35 heterodimers, or with a modified p40 polypeptide ornucleic acid, provide a very specific means to treat patients with p40deficiency.

A composition comprising a polypeptide or a nucleic acid of theinvention may also be used to treat conditions resulting from hyper- orneo-vascularization, such as age-related macular degeneration ordiabetic retinopathy, and to inhibit tumor growth.

An antagonist of the cellular receptor of the p40/p35 heterodimer (suchas, for example, a human interleukin-12 receptor), said antagonistcomprising a polypeptide of the invention or a fragment thereof, isuseful in situations where downregulation of T_(H)1-mediated responsesare desired. Such antagonist may include, but is not limited to, a p40homodimer comprising at least one modified p40 polypeptide of theinvention. In vitro and in vivo studies in mice have demonstrated thathomodimers of the murine p40 polypeptide bind to mouse receptor (withaffinity comparable to that of the heterodimer) and downregulate T_(H)1mediated responses (Ling, et al. (1994) J. Immunol 154:116-127 andGately, et al. (1996) Ann. NY Acad. Sci. 795:1-12). The p40 homodimerwas found to prevent induction and progression of ExperimentalAutoimmune Encephalomyelitis (EAE) in mice (Gately, et al. (1998) Annu.Rev. Immunol 16:495-521), an animal model for multiple sclerosis (MS) inhumans. Another antagonist contemplated by the invention comprises amodified p35 polypeptide of the invention plus the Epstein-Barr virusinduced gene 3 protein (EBI3). EBI3, which is structurally related tothe p40 polypeptide, has been shown to associate with p35 polypeptide;the product of this association appears to antagonize the biologicaleffects of interleukin-12 (Devergne, O. et al. (1997) Proc. Natl. Acad.Sci. USA 94:12041-12046). An antagonist comprising a polypeptide of theinvention may be administered to decrease cell-mediated immune response,in the treatment of, for example, autoimmune diseases such as MS, type Idiabetes mellitus, myasthenia gravis, rheumatoid arthritis, and systemiclupus erythematosus.

Modified p40 nucleic acids and/or modified p35 nucleic acids of theinvention appear to effect enhanced production of biologically activeheterodimers in vivo. This observed production enhancement may providefor more efficacious treatment with reduced toxicity or side-effects.Anti-tumor effects of interleukin-12 are known to be dose-dependent andrequire high localized concentrations for maximal efficacy (TrinchieriG. and Scott P. (1999) Curr. Top. Microbiol. Immunol. 238:57-78).Furthermore, genetic delivery of interleukin-12 nucleic acids results inless pronounced toxicity compared to systemic administration ofinterleukin-12 protein (Sun, Y. et al. (1998) Gene Ther. 5(4):481-90;Rakhmilevich A. L. et al. (1999) J. Immunother. 22(2):135-44).Therefore, modified p40 nucleic acids and/or modified p35 nucleic acidsof the invention that are capable of producing higher levels ofbiologically active heterodimeric cytokine in vivo than thecorresponding wild-type nucleic acids may be useful, e.g., as antitumoragents, as a vaccine adjuvant, or as replacement therapy forinterleukin-12 deficiency. In some instances, genetic delivery ofmodified p40 nucleic acids and/or modified p35 nucleic acids of theinvention may be a preferred route of administration (intra-corneal;ARMD) and potentially less cumbersome—especially if less injectionsprovide more sustained controlled release of biologically activeheterodimer.

Therapeutic and Prophylactic Compositions

Therapeutic or prophylactic compositions comprising one or more modifiedp40 or modified p35 polypeptide or nucleic acid of the invention aretested in appropriate in vitro, ex vivo, and in vivo animal models ofdisease, to confirm efficacy, tissue metabolism, and to estimatedosages, according to methods well known in the art. In particular,dosages can be determined by activity comparison of the modified p35 andmodified p40 polypeptides and nucleic acids to existing p35 andp40-based therapeutics or prophylactics, i.e., in a relevant assay. Inone aspect, the invention provides methods comprising administering oneor more modified p35 and/or modified p40 nucleic acids or polypeptidesof the invention (or fragments thereof) described above to a mammal,including, e.g., a human, primate, mouse, pig, dog, cat, cow, goat,rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalianvertebrate such as a bird (e.g., a chicken or duck) or a fish, orinvertebrate, as described in greater detail below. Such compositionstypically comprise one or more modified p35 and/or modified p40 nucleicacids or polypeptides of the invention (or fragments thereof) and anexcipient, including, e.g., a pharmaceutically acceptable excipient.

In one aspect, a composition of the invention is produced by digestingone or more nucleic acids of the invention (or fragments thereof) with arestriction endonuclease, an RNase, or a DNase.

In another aspect of the invention, compositions produced by incubatingone or more nucleic acids described above in the presence ofdeoxyribonucelotide triphosphates and a nucleic acid polymerase, e.g., athermostable polymerase, are provided.

The invention also includes compositions comprising two or more nucleicacids described above. The composition may comprise a library of nucleicacids, where the library contains at least about 5, 10, 20, 50, 100,150, or 200 or more such nucleic acids.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The modifiedp35 and/or modified p40 nucleic acids or polypeptides of the inventionare administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch modified p35 and/or modified p40 nucleic acids or polypeptides inthe context of the present invention to a patient are available, andalthough more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered for any of theprophylactic, therapeutic, and diagnostic methods described herein by anumber of routes including, but not limited to oral, intravenous,intraperitoneal, intramuscular, transdermal, subcutaneous, topical,sublingual, or rectal means, or by inhalation. Modified p35 and/ormodified p40 polypeptide compositions can also be administered vialiposomes. Such administration routes and appropriate formulations aregenerally known to those of skill in the art.

The modified p35 and/or modified p40 polypeptide or nucleic acid of theinvention, alone or in combination with other suitable components, canalso be made into aerosol formulations (i.e., they can be “nebulized”)to be administered via inhalation. Aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for existing p35 and/or p40 therapeutics orprophylactics, along with formulations in current use, are preferredroutes of administration and formulation for the modified p35 and/ormodified p40 polypeptide or nucleic acid of the invention (see, e.g.,Gollob J. A. et al. (2000) Clin. Cancer Res. 6:1678-1692).

Cells transduced with modified p35 and/or modified p40 nucleic acids asdescribed above in the context of ex vivo or in vivo therapy can also beadministered intravenously or parenterally as described above. It willbe appreciated that the delivery of cells to patients (e.g., humanpatients) is routine, e.g., delivery of cells to the blood viaintravenous or intraperitoneal administration.

The dose of modified p35 and/or modified p40 polypeptide or nucleic acidof the invention administered to a subject (e.g., patient), in thecontext of the present invention is sufficient to effect a beneficialtherapeutic or prophylactic response in the patient over time, or toinhibit infection by a pathogen, depending on the application. The dosewill be determined by the efficacy of the particular vector, orformulation, and the activity of the modified p35 and/or modified p40employed and the condition of the patient, as well as the body weight orsurface area of the patient to be treated. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,formulation, transduced cell type or the like in a particular patient.

In the therapeutic and prophylactic treatment methods of the inventiondescribed herein, an effective amount of a modified p40 and/or amodified p35 nucleic acid (e.g., DNA or mRNA) of the invention (e.g.,nucleic acid dosage) will generally be in the range of, e.g., from about0.05 microgram/kilogram (kg) to about 50 mg/kg, usually about 0.005-5mg/kg. However, as will be understood, the effective amount of thenucleic acid (e.g., nucleic acid dosage) and/or polypeptide (e.g.,polypeptide dosage) will vary in a manner apparent to those of ordinaryskill in the art according to a number of factors, including theactivity or potency of the polypeptide, the activity or potency of anynucleic acid construct (e.g., vector, promoter, expression system) to beadministered, the disease or condition (e.g., particular cancer) to betreated, and the subject to which or whom the nucleic acid is delivered.

For delivery of some polypeptides, e.g., by delivering nucleic acidsencoding such polypeptides, for example, adequate levels of translationand/or expression are achieved with a nucleic acid dosage of, e.g.,about 0.005 mg/kg to about 5 mg/kg. Dosages for other polypeptides (andnucleic acids encoding them) having a known biological activity can bereadily determined by those of skill in the art according to the factorsnoted above. Dosages used for other known p40 and/or p35 polypeptides(and nucleic acids encoding them) for particular diseases provideguidelines for determining dosage and treatment regimen for a nucleicacid or polypeptide of the invention. An effective amount of aheterodimeric p40/p35 polypeptide, comprising a modified p40 polypeptideand/or a modified p35 polypeptide of the invention, may be in the rangeof from about 1 nanogram (ng)/kg to about 1 mg/kg, and more typicallyfrom about 10 ng/kg to about 500 ng/kg (Gollob J. A. et al., supra.)

A composition for use in therapeutic and prophylactic treatment methodsof the invention described herein may comprise, e.g., a concentration ofa modified p40 nucleic acid and/or a modified p35 nucleic acid (e.g.,DNA or mRNA) of the invention of from about 0.1 microgram/milliliter(ml) to about 20 mg/ml and a pharmaceutically acceptable carrier (e.g.,aqueous carrier).

A composition for use in therapeutic and prophylactic treatment methodsof the invention described herein may comprise, e.g., a concentration ofa modified p40 polypeptide and/or a modified p35 polypeptide of theinvention in an amount as described above and herein and apharmaceutically acceptable carrier (e.g., aqueous carrier).

In determining the effective amount of the vector, cell type, orformulation to be administered in the treatment or prophylaxis of e.g.,tumors, or infectious agents, such as bacterial, protozoal,intracellular parasitic, and viral infections, the physician evaluatescirculating plasma levels, vector/cell/formulation/toxicities of themodified p35 and/or modified p40 nucleic acid or polypeptide,progression of the disease, and the production of anti-vector and/oranti-(modified p35 and/or modified p40) antibodies.

The dose administered, e.g., to a 70 kilogram patient will be in therange equivalent to dosages of currently-used p40/p35 therapeutic orprophylactic proteins, and doses of vectors or cells which producemodified p35 and/or modified p40 polypeptide sequences are calculated toyield an equivalent amount of modified p35 and/or modified p40 nucleicacid or expressed protein. The vectors of this invention can supplementtreatment of e.g., tumors, or infectious agents, such as bacterial,protozoal, intracellular parasitic, and viral infections by any knownconventional therapy, including cytotoxic agents, nucleotide analogues(e.g., when used for treatment of HIV infection), biologic responsemodifiers, and the like.

For administration, modified p40 and/or modified p35 polypeptides,nucleic acids, vectors, and transduced cells of the present inventioncan be administered at a rate determined by the LD-50 of the modifiedp40 and/or modified p35 polypeptide, vector, or transduced cell type,and the side-effects of the modified p40 and/or modified p35polypeptide, vector or cell type at various concentrations, as appliedto the mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

For introduction of recombinant modified p35 and/or modified p40 nucleicacid transduced cells into a subject (e.g., patient), blood samples areobtained prior to infusion, and saved for analysis. Between 1×10⁶ and1×10¹² transduced cells are infused intravenously over 60-200 minutes.Vital signs and oxygen saturation by pulse oximetry are closelymonitored. Blood samples are obtained 5 minutes and 1 hour followinginfusion and saved for subsequent analysis. Leukopheresis, transductionand reinfusion are optionally repeated every 2 to 3 months for a totalof 4 to 6 treatments in a one year period. After the first treatment,infusions can be performed on a outpatient basis at the discretion ofthe clinician. If the reinfusion is given as an outpatient, theparticipant is monitored for at least 4, and preferably 8 hoursfollowing the therapy. Transduced cells are prepared for reinfusionaccording to established methods. See, Abrahamsen et al. (1991) J. Clin.Apheresis 6:48-53; Carter et al. (1988) J. Clin. Arpheresis 4:113-117;Aebersold et al. (1988), J. Immunol. Methods 112: 1-7; Muul et al.(1987) J. Immunol. Methods 101:171-181 and Carter et al. (1987)Transfusion 27:362-365. After a period of about 2-4 weeks in culture,the cells should number between 1×10⁶ and 1×10¹². In this regard, thegrowth characteristics of cells vary from patient to patient and fromcell type to cell type. About 72 hours prior to reinfusion of thetransduced cells, an aliquot is taken for analysis of phenotype, andpercentage of cells expressing the therapeutic or prophylactic agent.

If a subject (e.g., patient) undergoing infusion of a vector ortransduced cell or protein formulation develops fevers, chills, ormuscle aches, he/she receives the appropriate dose of aspirin,ibuprofen, acetaminophen or other pain/fever controlling drug. Subjects(e.g., patients) who experience reactions to the infusion such as fever,muscle aches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistamines. Cell infusion isslowed or discontinued depending upon the severity of the reaction.

Therapeutic and Prophylactic Treatment Methods

The present invention also includes methods of therapeutically orprophylactically treating a disease or disorder by administering in vivoor ex vivo one or more nucleic acids or polypeptides of the inventiondescribed above (or compositions comprising a pharmaceuticallyacceptable excipient and one or more such nucleic acids or polypeptides)to a subject, including, e.g., a mammal, including, e.g., a human,primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse,sheep; or a non-mammalian vertebrate such as a bird (e.g., a chicken orduck) or a fish, or invertebrate.

In one aspect of the invention, in ex vivo methods, one or more cells ora population of cells of interest of the subject (e.g., tumor cells,tumor tissue sample, organ cells, blood cells, cells of the skin, lung,heart, muscle, brain, mucosae, liver, intestine, spleen, stomach,lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) areobtained or removed from the subject and contacted with an amount of apolypeptide of the invention that is effective in prophylactically ortherapeutically treating the disease, disorder, or other condition. Thecontacted cells are then returned or delivered to the subject to thesite from which they were obtained or to another site (e.g., includingthose defined above) of interest in the subject to be treated. Ifdesired, the contacted cells may be grafted onto a tissue, organ, orsystem site (including all described above) of interest in the subjectusing standard and well-known grafting techniques or, e.g., delivered tothe blood or lymph system using standard delivery or transfusiontechniques.

The invention also provides in vivo methods in which one or more cellsor a population of cells of interest of the subject are contacteddirectly or indirectly with an amount of a polypeptide of the inventioneffective in prophylactically or therapeutically treating the disease,disorder, or other condition. In direct contact/administration formats,the polypeptide is typically administered or transferred directly to thecells to be treated or to the tissue site of interest (e.g., tumorcells, tumor tissue sample, organ cells, blood cells, cells of the skin,lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach,lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) by anyof a variety of formats, including topical administration, injection(e.g., by using a needle or syringe), or vaccine or gene gun delivery,pushing into a tissue, organ, or skin site. The polypeptide can bedelivered, for example, intramuscularly, intradermally, subdermally,subcutaneously, orally, intraperitoneally, intrathecally, intravenously,or placed within a cavity of the body (including, e.g., during surgery),or by inhalation or vaginal or rectal administration.

In in vivo indirect contact/administration formats, the polypeptide istypically administered or transferred indirectly to the cells to betreated or to the tissue site of interest, including those describedabove (such as, e.g., skin cells, organ systems, lymphatic system, orblood cell system, etc.), by contacting or administering the polypeptideof the invention directly to one or more cells or population of cellsfrom which treatment can be facilitated. For example, tumor cells withinthe body of the subject can be treated by contacting cells of the bloodor lymphatic system, skin, or an organ with a sufficient amount of thepolypeptide such that delivery of the polypeptide to the site ofinterest (e.g., tissue, organ, or cells of interest or blood orlymphatic system within the body) occurs and effective prophylactic ortherapeutic treatment results. Such contact, administration, or transferis typically made by using one or more of the routes or modes ofadministration described above.

In another aspect, the invention provides ex vivo methods in which oneor more cells of interest or a population of cells of interest of thesubject (e.g., tumor cells, tumor tissue sample, organ cells, bloodcells, cells of the skin, lung, heart, muscle, brain, mucosae, liver,intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate,mouth, tongue, etc.) are obtained or removed from the subject andtransformed by contacting said one or more cells or population of cellswith a polynucleotide construct comprising a target nucleic acidsequence of the invention that encodes a biologically active polypeptideof interest (e.g., a polypeptide of the invention) that is effective inprophylactically or therapeutically treating the disease, disorder, orother condition. The one or more cells or population of cells iscontacted with a sufficient amount of the polynucleotide construct and apromoter controlling expression of said nucleic acid sequence such thatuptake of the polynucleotide construct (and promoter) into the cell(s)occurs and sufficient expression of the target nucleic acid sequence ofthe invention results to produce an amount of the biologically activepolypeptide effective to prophylactically or therapeutically treat thedisease, disorder, or condition. The polynucleotide construct mayinclude a promoter sequence (e.g., CMV promoter sequence) that controlsexpression of the nucleic acid sequence of the invention and/or, ifdesired, one or more additional nucleotide sequences encoding at leastone or more of another polypeptide of the invention, a cytokine,adjuvant, or co-stimulatory molecule, or other polypeptide of interest.

Following transfection, the transformed cells are returned, delivered,or transferred to the subject to the tissue site or system from whichthey were obtained or to another site (e.g., tumor cells, tumor tissuesample, organ cells, blood cells, cells of the skin, lung, heart,muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphaticsystem, cervix, vagina, prostate, mouth, tongue, etc.) to be treated inthe subject. If desired, the cells may be grafted onto a tissue, skin,organ, or body system of interest in the subject using standard andwell-known grafting techniques or delivered to the blood or lymphaticsystem using standard delivery or transfusion techniques. Such delivery,administration, or transfer of transformed cells is typically made byusing one or more of the routes or modes of administration describedabove. Expression of the target nucleic acid occurs naturally or can beinduced (as described in greater detail below) and an amount of theencoded polypeptide is expressed sufficient and effective to treat thedisease or condition at the site or tissue system.

In another aspect, the invention provides in vivo methods in which oneor more cells of interest or a population of cells of the subject (e.g.,including those cells and cells systems and subjects described above)are transformed in the body of the subject by contacting the cell(s) orpopulation of cells with (or administering or transferring to thecell(s) or population of cells using one or more of the routes or modesof administration described above) a polynucleotide construct comprisinga nucleic acid sequence of the invention that encodes a biologicallyactive polypeptide of interest (e.g., a polypeptide of the invention)that is effective in prophylactically or therapeutically treating thedisease, disorder, or other condition.

The polynucleotide construct can be directly administered or transferredto cell(s) suffering from the disease or disorder (e.g., by directcontact using one or more of the routes or modes of administrationdescribed above). Alternatively, the polynucleotide construct can beindirectly administered or transferred to cell(s) suffering from thedisease or disorder by first directly contacting non-diseased cell(s) orother diseased cells using one or more of the routes or modes ofadministration described above with a sufficient amount of thepolynucleotide construct comprising the nucleic acid sequence encodingthe biologically active polypeptide, and a promoter controllingexpression of the nucleic acid sequence, such that uptake of thepolynucleotide construct (and promoter) into the cell(s) occurs andsufficient expression of the nucleic acid sequence of the inventionresults to produce an amount of the biologically active polypeptideeffective to prophylactically or therapeutically treat the disease ordisorder, and whereby the polynucleotide construct or the resultingexpressed polypeptide is transferred naturally or automatically from theinitial delivery site, system, tissue or organ of the subject's body tothe diseased site, tissue, organ or system of the subject's body (e.g.,via the blood or lymphatic system). Expression of the target nucleicacid occurs naturally or can be induced (as described in greater detailbelow) such that an amount of the encoded polypeptide is expressedsufficient and effective to treat the disease or condition at the siteor tissue system. The polynucleotide construct may include a promotersequence (e.g., CMV promoter sequence) that controls expression of thenucleic acid sequence and/or, if desired, one or more additionalnucleotide sequences encoding at least one or more of anotherpolypeptide of the invention, a cytokine, adjuvant, or co-stimulatorymolecule, or other polypeptide of interest.

In each of the in vivo and ex vivo treatment methods described above, acomposition comprising an excipient and the polypeptide or nucleic acidof the invention can be administered or delivered. In one aspect, acomposition comprising a pharmaceutically acceptable excipient and apolypeptide or nucleic acid of the invention is administered ordelivered to the subject as described above in an amount effective totreat the disease or disorder.

In another aspect, in each in vivo and ex vivo treatment methoddescribed above, the amount of polynucleotide administered to thecell(s) or subject can be an amount sufficient that uptake of saidpolynucleotide into one or more cells of the subject occurs andsufficient expression of said nucleic acid sequence results to producean amount of a biologically active polypeptide effective to enhance animmune response in the subject, including an immune response induced byan immunogen (e.g., antigen). In another aspect, for each such method,the amount of polypeptide administered to cell(s) or subject can be anamount sufficient to enhance an immune response in the subject,including that induced by an immunogen (e.g., antigen).

In yet another aspect, in an in vivo or in vivo treatment method inwhich a polynucleotide construct (or composition comprising apolynucleotide construct) is used to deliver a physiologically activepolypeptide to a subject, the expression of the polynucleotide constructcan be induced by using an inducible on- and off-gene expression system.Examples of such on- and off-gene expression systems include the Tet-On™Gene Expression System and Tet-Off™ Gene Expression System (see, e.g.,Clontech Catalog 2000, pg. 110-111 for a detailed description of eachsuch system), respectively. Other controllable or inducible on- andoff-gene expression systems are known to those of ordinary skill in theart. With such system, expression of the target nucleic of thepolynucleotide construct can be regulated in a precise, reversible, andquantitative manner. Gene expression of the target nucleic acid can beinduced, for example, after the stable transfected cells containing thepolynucleotide construct comprising the target nucleic acid aredelivered or transferred to or made to contact the tissue site, organ orsystem of interest. Such systems are of particular benefit in treatmentmethods and formats in which it is advantageous to delay or preciselycontrol expression of the target nucleic acid (e.g., to allow time forcompletion of surgery and/or healing following surgery; to allow timefor the polynucleotide construct comprising the target nucleic acid toreach the site, cells, system, or tissue to be treated; to allow timefor the graft containing cells transformed with the construct to becomeincorporated into the tissue or organ onto or into which it has beenspliced or attached, etc.)

Integrated Systems

The present invention provides computers, computer readable media andintegrated systems comprising character strings corresponding to thesequence information herein for the polypeptides and nucleic acidsherein, including, e.g., those sequences listed herein and the varioussilent substitutions and conservative substitutions thereof.

Various methods and genetic algorithms (GOs) known in the art can beused to detect homology or similarity between different characterstrings, or can be used to perform other desirable functions such as tocontrol output files, provide the basis for making presentations ofinformation including the sequences and the like. Examples includeBLAST, discussed supra.

Thus, different types of homology and similarity of various stringencyand length can be detected and recognized in the integrated systemsherein. For example, many homology determination methods have beendesigned for comparative analysis of sequences of biopolymers, forspell-checking in word processing, and for data retrieval from variousdatabases. With an understanding of double-helix pair-wise complementinteractions among 4 principal nucleobases in natural polynucleotides,models that simulate annealing of complementary homologouspolynucleotide strings can also be used as a foundation of sequencealignment or other operations typically performed on the characterstrings corresponding to the sequences herein (e.g., word-processingmanipulations, construction of figures comprising sequence orsubsequence character strings, output tables, etc.). An example of asoftware package with GOs for calculating sequence similarity is BLAST,which can be adapted to the present invention by inputting characterstrings corresponding to the sequences herein.

Similarly, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can be adapted to the present invention by inputting acharacter string corresponding to the modified p35 sequences and/ormodified p40 sequences of the invention (either nucleic acids orpolypeptides, or both). For example, the integrated systems can includethe foregoing software having the appropriate character stringinformation, e.g., used in conjunction with a user interface (e.g., aGUI in a standard operating system such as a Windows, Macintosh or LINUXsystem) to manipulate strings of characters. As noted, specializedalignment programs such as BLAST can also be incorporated into thesystems of the invention for alignment of nucleic acids or proteins (orcorresponding character strings).

Integrated systems for analysis in the present invention typicallyinclude a digital computer with GO software for aligning sequences, aswell as data sets entered into the software system comprising any of thesequences herein. The computer can be, e.g., a PC (Intel ×86 or Pentiumchip-compatible DOS™, OS2™ WINDOWS™ WINDOWS NT™, WINDOWS95™, WINDOWS98™LINUX based machine, a MACINTOSH™, Power PC, or a UNIX based (e.g., SUN™work station) machine) or other commercially common computer which isknown to one of skill. Software for aligning or otherwise manipulatingsequences is available, or can easily be constructed by one of skillusing a standard programming language such as Visualbasic, Fortran,Basic, Java, or the like.

Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation.

The software can also include output elements for controlling nucleicacid synthesis (e.g., based upon a sequence or an alignment of asequences herein) or other operations which occur downstream from analignment or other operation performed using a character stringcorresponding to a sequence herein.

In one embodiment, the invention provides an integrated systemcomprising a computer or computer readable medium comprising a databasehaving one or more sequence records. Each of the sequence recordscomprises one or more character strings corresponding to a nucleic acidor polypeptide or protein sequence selected from SEQ ID NO:1 to SEQ IDNO:14, SEQ ID NO:16 to SEQ ID NO:35, SEQ ID NO:39, and SEQ ID NO:40. Theintegrated system further comprises a use input interface allowing a useto selectively view the one or more sequence records. In one suchintegrated system, the computer or computer readable medium comprises analignment instruction set that aligns the character strings with one ormore additional character strings corresponding to a nucleic acid orpolypeptide or protein sequence.

One such integrated system includes an instruction set that comprises atleast one of the following: a local homology comparison determination, ahomology alignment determination, a search for similarity determination,and a BLAST determination. In some embodiments, the system furthercomprises a readable output element that displays an alignment producedby the alignment instruction set. In another embodiment, the computer orcomputer readable medium further comprises an instruction set thattranslates at least one nucleic acid sequence which comprises a sequenceselected from SEQ ID NO:1 to SEQ ID NO:7 or SEQ ID NO:16 to SEQ ID NO:25into an amino acid sequence. The instruction set may select the nucleicacid by applying a codon usage instruction set or an instruction setwhich determines sequence identity to a test nucleic acid sequence.

Methods of using a computer system to present information pertaining toat least one of a plurality of sequence records stored in a database arealso provided. Each of the sequence records comprises at least onecharacter string corresponding to SEQ ID NO:1 to SEQ ID NO:14, SEQ IDNO:16 to SEQ ID NO:35, SEQ ID NO:39, and SEQ ID NO:40. The methodcomprises determining at least one character string corresponding to oneor more of SEQ ID NO:1 to SEQ ID NO:14, SEQ ID NO:16 to SEQ ID NO:35,SEQ ID NO:39, and SEQ ID NO:40 or a subsequence thereof; determiningwhich of the at least one character string of the list are selected by auser; and displaying each of the selected character strings, or aligningeach of the selected character strings with an additional characterstring. The method may further comprise displaying an alignment of eachof the selected character strings with an additional character stringand/or displaying the list.

Kits

In an additional aspect, the present invention provides kits embodyingthe methods, composition, systems and apparatus herein. Kits of theinvention optionally comprise one or more of the following: (1) anapparatus, system, system component or apparatus component as describedherein; (2) instructions for practicing the methods described herein,and/or for operating the apparatus or apparatus components herein and/orfor using the compositions herein; (3) one or more modified p35 and/ormodified p40 composition (such as e.g., compositions comprising at leastone modified p35 and/or modified p40 nucleic acid or polypeptide orfragment thereof, cell, vector, etc., of the invention) or component(modified p35 and/or modified p40 nucleic acid or polypeptide orfragment thereof, cell, vector, etc., of the invention); (4) a containerfor holding one or more aspects of the invention, including suchcomponents or compositions, and (5) packaging materials.

In a further aspect, the present invention provides for the use of anyapparatus, apparatus component, composition or kit herein, for thepractice of any method or assay herein, and/or for the use of anyapparatus or kit to practice any assay or method herein.

EXAMPLES Example I Preparation and Screening of Polypeptides

Nucleic acids encoding polypeptides of the invention, and nucleic acidsencoding wt-p40 and wt-p35 polypeptides, were individually subclonedinto the pcDNA1.3 (+) (Invitrogen) expression/secretion vector andtransformed into E. Coli. Transformants were propagated in 96-wellblocks, and plasmid DNA was purified using an automated DNA purificationsystem (Qiagen). Plasmids were transfected into COS-7 or 293 mammaliancells using a high-throughput 96-well format and Superfect Reagent(Qiagen). The culture medium was harvested and analyzed for proteinexpression and biological activity after a 48-72 hour incubation period.

In some instances, sequences of naturally-occurring (also referred toherein as wild-type or “wt”) p35 nucleic acids and modified p35 nucleicacids were further modified to encode an EHtag (SEQ ID NO:44) at theC-terminus to facilitate purification and quantitation of the ˜70 kDaheterodimers (comprising a p35 polypeptide and a p40 polypeptide).

Quantitation of Expression and Purification of Heterodimeric Proteins

Protein concentration was estimated by analyzing culture supernatants,or purified heterodimeric proteins, on a 4-12% NuPAGE gel (Novex, SanDiego, Calif.) followed by either staining with SilverXpress stainingkit (Novex, San Diego, Calif.) or chemiluminescent detection usinganti-p35 mAB (R&D Systems, Pharmingen) or a mouse anti-polyhistidine tagmAB (Accurate, Westbury, N.Y.), followed by goat anti-mouse IgGconjugated to HRP (Pharmacia-Amersham, Piscataway, N.J.). Alternatively,concentration of purified protein was estimated by absorbance at 280 nm.

Large-scale transfections of human 293 cells were performed in 150millimeter (mm) dishes with Superfect Reagent (Qiagen, Valencia, Calif.)using 10 microgram (ug) of each wt or modified p35-EHtag and p40 subunitexpression vectors. Culture supernatant was harvested 72 hourspost-transfection and centrifuged at low speed followed by filtrationthrough a 0.2 micron (u) filter (Nalgene, Rochester, N.Y.) to removecellular debris. The centrifuged supernatant was passed through ananti-E affinity column (Pharmacia-Amersham, Piscataway, N.J.)equilibrated in 20 milliMolar (mM) phosphate buffer pH 7.0 and proteineluted with 0.1 Molar (M) glycine pH 3.0 followed by neutralization with1/10 volume of 1 M Tris pH 8.2. The sample was then buffered exchangedinto phosphate-buffered saline (PBS) and concentrated with Centriconplus-80 filter devices (Millipore, Bedford, Mass.).

T-Cell Proliferation Assay

The assay was performed as described by Punnonen and de Vries (1994;Journal of Immunology 152: 1094). Human peripheral blood leukocytes(PBLs) were isolated from buffy coats by density-gradient centrifugationusing Histopaque (Sigma), washed twice with PBS, and resuspended at aconcentration of 2×10⁶ cells/ml in RPMI medium (Gibco-BRL) supplementedwith 10% fetal calf serum (Hyclone), 1× glutamine (Gibco-BRL) and 100ug/ml of penicillin and streptomycin (Gibco-BRL). The isolated PBLs werethen cultured in T75 flasks for 96 hours in the presence of 5 ug/mlphytohemagglutinin (PHA; Sigma) to induce activation of T-lymphocytes.Subsequently, the cells were washed twice with PBS and adjusted to 4×10⁵cells/ml in RPMI medium.

Assays were performed either (a) directly on serial dilutions of culturesupernatants from transfected mammalian cells, or (b) on proteinspurified from culture supernatants. Serial dilutions of expressionculture medium, or of partially purified protein, were placed in 96-wellround bottom plates (Costar). A 100 microliter (ul) aliquot ofresuspended T-cells, activated as described above, was immediately addedto each well with an automated multi-channel pipet (Matrix) and theplates were incubated at 37° C. with 5% CO₂ for 48 hours. This wasfollowed by an additional incubation period of 16 hours in the presenceof 1 uCi of ³H-thymidine (Amersham). The cells were then harvested andthe amount of 3H-thymidine incorporation—a measure of cytokine-dependentT_(H)1 proliferation was measured using a 1450 micro-beta Trilux liquidscintillation counter (Wallac).

T_(H)1 Differentiation/Interferon-γ Induction Assay

Production of a human T_(H)1 specific cytokine, interferon-γ, wasmeasured using a modification of the method described by Murphy et al.(1985; Journal of Experimental Medicine 164:263). Human T-cells werepurified by negative selection using antibodies to CD14, CD19, CD56 andCD16 (Beckton-Dickinson). The homogenous population of T-cells wascultured at 1×10⁶ cells/well for 5 days in Iscove medium, in thepresence or absence of test proteins. Before activating the cells withsoluble anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml), the cells wereharvested and washed with PBS. The cells were incubated at 37° C. with5% CO2 for 48 hours prior to harvesting the conditioned medium andmeasuring the level of interferon-γ with a commercially available ELISAkit (R&D Systems).

Results

Culture media from mammalian cells expressing-nucleic acids of theinvention, and purified heterodimeric proteins, were tested for T-cellproliferative activity and interferon-γ induction activity as describedabove. Representative results are provided as follows. Nucleic acidsencoding modified p40 polypeptides of the invention were co-expressedwith nucleic acids encoding a wt-p35 polypeptide (SEQ ID NO:36). Ingeneral, significantly greater T-cell proliferative activities wereobserved in the culture media of cells expressing and secretingheterodimers comprising modified p40 polypeptides of the invention thanwere observed in the culture media of control cells expressing andsecreting heterodimers formed of wt-p40 and wt-p35 polypeptides.

FIG. 2 shows that co-expression of a modified p40 nucleic acid of theinvention, (SEQ ID NO:7 encoding R16-51), with nucleic acid encoding awt-p35 polypeptide (SEQ ID NO:36) in COS-7 cells, results in secretionof active protein into the conditioned media. Relative T-cellproliferative activity of the modified-p40/wt-p35 heterodimer comparedto that of the control wt-p40/wt-p35 heterodimer were estimated bycomparing the n-fold dilution of test culture media necessary to achievean equal level of activity to that of the control culture media. Assayof serial dilutions of the media showed that the modified p40/wt-p35heterodimer required about an 4-fold dilution of media to achieve alevel of ³H-thymidine incorporation (CPM) equivalent to that of awt-p40/wt-p35 heterodimer. This is indicative of about an 4-fold greaterT-cell proliferative activity of the expressed, secreted R16-51/wt-p35heterodimer over that of the control culture expressing and secretingwt-p40/wt-p35 heterodimer. FIG. 3 shows the relative T-cellproliferative activities of culture media dilutions of human 293 cellsco-expressing a nucleic acid encoding a wt-p35 polypeptide (SEQ IDNO:36) plus one of the following modified p40 nucleic acids of theinvention: SEQ ID NO:6 encoding A16-94; SEQ ID NO:2 encoding B8-96; orSEQ ID NO:1 encoding C2-22; or a nucleic acid (SEQ ID NO: 37) encoding awt-p40 polypeptide. Relative activities of the modified p40/wt-p35heterodimers compared to that of the control wt-p40/wt-p35 heterodimerwere estimated by comparing the n-fold dilution of test culture medianecessary to achieve an equal level of activity to that of the controlculture media. Assay of serial dilutions of the media showed that themodified p40/wt-p35 heterodimers comprising modified p40 polypeptidesA16-94 and B8-96 required at least about an 8-fold dilution and at leastabout a 16-fold dilution, respectively, of culture supernatant toachieve a level of ³H-thymidine incorporation (CPM) equivalent to thatof wt-p40/wt-p35 heterodimer, while the modified p40/wt-p35 heterodimercomprising modified p40 polypeptide C2-22 required at least about a 32-to 64-fold dilution of media to achieve a level of ³H-thymidineincorporation (CPM) equivalent to that of wt-p40/wt-p35 heterodimer (asindicated by the dashed horizontal line). In other words, culture mediaof the expressed and secreted A16-94/wt-p35 and B8-96/wt-p35heterodimers showed at least an 8-fold and at least a 16-fold greaterT-cell proliferative activity, respectively, and the culture media ofthe expressed and secreted C2-22/wt-p35 heterodimer showed at least 32-to 64-fold greater T-cell proliferative activity, than that of thecontrol culture media of the expressed and secreted wt-p40/wt-p35heterodimer.

FIGS. 4, 5, and 6, respectively, show the T-cell proliferativeactivities of culture media of cells co-expressing a nucleic acidencoding a naturally-occurring (wt-)p35 polypeptide (SEQ ID NO:36) plusone of the following modified p40 nucleic acids: SEQ ID NO:5 encodingA3-48 (FIG. 4), SEQ ID NO:3 encoding B2-52 (FIG. 5), and SEQ ID NO:4encoding B1-81 (FIG. 6), in comparison to dilutions of culture media ofcontrol cells co-expressing nucleic acids encoding naturally-occurringwt-p40/wt-p35 polypeptides. The results of FIGS. 4-6 are indicative ofat least about a 4- to 8-fold greater T-cell proliferative activity ofeach of the expressed, secreted modified p40/wt-p35 heterodimers overthat of the control expressed, secreted wt-p40/wt-p35 heterodimer.

FIG. 7 shows that COS-7 cells co-expressing nucleic acid encoding anexemplary modified p40 polypeptide of the invention plus nucleic acidencoding wt-p35 polypeptide consistently secreted more heterodimeric(˜70 kDa) protein into the culture media than COS-7 cells co-expressingnucleic acids encoding wt-p40 and wt-p35 polypeptides. Heterodimer (p70)production was quantitated by immunoblot analysis, using an anti-p35mAB, of equivalent volumes of crude culture medium from COS-7 cellsco-expressing nucleic acids encoding wt-p35 polypeptide (SEQ ID NO:36)plus either a nucleic acid encoding one of the following exemplarymodified p40 polypeptides, identified as A16-94 (SEQ ID NO:13), A3-48(SEQ ID NO:12), B1-81 (SEQ ID NO:11), B2-52 (SEQ ID NO:10), B8-96 (SEQID NO:9) or C2-22 (SEQ ID NO:8), or a nucleic acid encoding a wt-p40(SEQ ID NO:15). Thus, the increased proliferative activities of themodified heterodimers over the wild-type heterodimers may be in partattributable to enhanced production of modified heterodimers as comparedto the wild-type counterparts.

As noted above and in FIG. 3, culture media from cells expressing themodified p40 C2-22/wt-p35 heterodimer showed an up to 64-fold increasein relative proliferative activity over that of culture media cellsexpressing the wild-type heterodimer (that is, up to a 64-fold lowervolume of culture media from cells expressing the modified heterodimerwas required to achieve an activity value equivalent to that of a 1×volume of culture media from cells expressing the wild-typeheterodimer). To estimate the relative contributions of enhancedmodified heterodimer production versus increased activity to thisobserved activity increase, heterodimers were first quantitated byimmunoblot of equivalent dilutions of partially purified heterodimerfrom wt-p40/wt-p35 and C2-22/wt-p35 cultures using an anti-p35 mAB. Fromdensitometery analysis, an estimated 16-fold enhancement of expressionof C2-22/wt-p35 heterodimer over that of the control wt-p40/wt-p35heterodimer was observed (FIG. 8). Based on this quantitation,equivalent concentrations of partially purified C2-22/wt-p35 heterodimershowed an approximately four-fold higher proliferative activity comparedto partially purified control wt-p40/wt-p35 heterodimer in the humanT-cell proliferation assay after normalizing for protein concentration(FIG. 9). Taken together, these data suggest that the observed 32- to64-fold activity enhancement observed in the culture broth assay isconsistent with about a 16-fold increase in active heterodimerproduction, and about a four-fold higher proliferative activity of theC2-22/wt-p35 heterodimer over that of the control wild-type heterodimer.

The ability of partially purified C2-22/wt-p35 heterodimer to inducesynthesis and secretion of the T_(H)1 specific cytokine, interferon-γ,by human T-cells was determined in the human T_(H)1differentiation/IFN-γ induction assay. FIG. 10 shows the levels of humanIFN-γ produced by human T-cells incubated in the presence or absence ofpartially purified C2-22/wt-p35 or control wt-p40/wt-p35 heterodimers,followed by activation with anti-CD3 and anti-CD28. The amount of IFN-γproduced by human T-cells incubated with ˜1 ng/ml of partially purifiedp40C2-22/wt-p35 heterodimer was significantly greater than that of cellstreated with ˜1 ng/ml of partially purified control wt-p40/wt-p35heterodimer.

Nucleic acids encoding modified p35 polypeptides of the invention wereco-expressed with nucleic acids encoding either wt-p40 or variousmodified p40 polypeptides of the invention. In general, significantlygreater T-cell proliferative activities were observed in the culturemedia of cells expressing and secreting heterodimers comprising modifiedp35 polypeptides of the invention, and in the culture media of cellsexpressing and secreting heterodimers comprising both modified p35 andmodified p40 polypeptides of the inventions, over T-cell proliferativeactivities of the culture media of control cells expressing andsecreting wild-type heterodimers formed of naturally-occurring wt-p40and wt-p35 polypeptides.

A “fully modified” heterodimer C2-22/R2-571-EHtag, comprising a modifiedp40 polypeptide comprising a sequence identified as the maturepolypeptide region (amino acid residues 23-324) of SEQ ID NO:8, and amodified p35 polypeptide comprising a sequence identified as the maturepolypeptide region (amino acid residues 23-2.19) of SEQ ID NO:31 plusthe C-terminal EHtag, was purified and quantitated as described above.FIG. 12 shows that the T-cell proliferative activity of the purifiedfully modified heterodimer (C2-22/R2-571-EHtag) is about 8 times greaterthan that of purified control wild-type (wt-p40/wt-p35-EHtag)heterodimer, after normalizing for protein concentration. FIG. 13 showsthat human T-cells incubated with the purified “fully modified”heterodimer (C2-22/R2-571-EHtag) at a concentration of either ˜10 ng/mlor ˜50 ng/ml produced 3 to 5 times more IFN-γ than human T-cellsincubated with equal concentrations of purified control wild-type(wt-p40/wt-p35-EHtag) heterodimer.

FIG. 14 shows a western blot, probed using an anti-E tag monoclonalantibody, of purified heterodimers produced by co-expressing in 293cells (a) a modified p40 nucleic acid of the invention (comprising theC2-22 polynucleotide sequence SEQ ID NO:1) plus a modified p35 nucleicacid of the invention (comprising the R2-571 polynucleotide sequence SEQID NO:21), and (b) wt-p40 and wt-p35 nucleic acids (comprising thepolynucleotide sequences SEQ ID NO:37 and SEQ ID NO:38, respectively).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques, methods, compositions,apparatus and systems described above may be used in variouscombinations. All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

1. An isolated or recombinant nucleic acid, comprising a polynucleotidesequence selected from the group consisting of: (a) a polynucleotidesequence comprising the mature polypeptide coding region of a sequenceselected from SEQ ID NO:1 to SEQ ID NO:7, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide, the polypeptide comprising an amino acid sequence havingat least 90% amino acid sequence identity to the mature polypeptideregion of a sequence selected from SEQ ID NO:8 to SEQ ID NO:14, or acomplementary polynucleotide sequence thereof; (c) a polynucleotidesequence which hybridizes under highly stringent conditions oversubstantially the entire length of polynucleotide sequence (a) or (b);(d) a polynucleotide sequence comprising a fragment of (a), (b), or (c),which fragment encodes a polypeptide having T-cell proliferativeactivity in the presence of a p35 polypeptide.
 2. The nucleic acid ofclaim 1, wherein the nucleic acid encodes a polypeptide having T-cellproliferative activity in the presence of a p35 polypeptide, the p35polypeptide comprising a sequence selected from a mature polypeptideregion of a wild-type p35 polypeptide and a mature polypeptide region ofa modified p35 polypeptide.
 3. The nucleic acid of claim 2, wherein thep35 polypeptide comprises a sequence comprising amino acid residues 23to 219 of SEQ ID NO:36.
 4. The nucleic acid of claim 3, wherein the p35polypeptide comprises a sequence comprising amino acid residues 23 to219 of SEQ ID NO:31 or SEQ ID NO:32.
 5. An isolated or recombinantnucleic acid, comprising a polynucleotide sequence encoding a modifiedp40 polypeptide comprising a modification at an equivalent position tothat in the amino acid sequence of a human p40 polypeptide (SEQ IDNO:15), the modification selected from the group consisting of: (a) asubstitution of the specified amino acid for a different amino acid atone or more equivalent position selected from Leu62, Ser71, Gln78,His99, Thr127, Arg130, Lys185, Glu186, Tyr187, Glu188, Ser190, Asp196,Met211, Val289, Ser305, Ser307, Arg309, and Gln311; (b) a deletion ofequivalent amino acid residues Arg181 to Asn184 inclusive, or asubstitution, of equivalent amino acid residues Arg181 to Asn184inclusive, for the amino acids Ser-(Leu or Met)-(Glu or Asp)-His-Arg;(c) a deletion of equivalent amino acid residues Asp287 and Arg288; and(d) at least two of (a), (b), or (c); wherein the numbering of aminoacid residue positions corresponds to that of SEQ ID NO:15.
 6. Thenucleic acid of claim 5, wherein the modified p40 polypeptide is amodification of a p40 polypeptide selected from the group consisting ofp40 polypeptides encoded by nucleic acids having the GenBank accessionnumbers: M65272, M65290, U19841, U19834, Y11129, U83184, Y07762,AF054607, U49100, AF091134, U57752, U10160, AF007576, AF004024, U11815,U08317, X97019, AF082494, AF133197, U16674, M86671, S82426, AF097507,and AF046211.
 7. The nucleic acid of claim 5, wherein the modified p40polypeptide is a modification of a human p40 polypeptide comprisingamino acid residues 23 to 328 of SEQ ID NO:15.
 8. The nucleic acid ofclaim 5, wherein the modified p40 polypeptide comprises one or moresubstitution selected from the group consisting of: Leu62Ser; Ser71Thr;Gln78His; His99(Arg or Gln); Thr127(Ser or Ile); Arg130Lys; Lys185Glu;Glu186Tyr; Tyr187(Lys or Asn); Glu188Lys; Ser190(Arg or Thr); Asp196Gly;Met211Val; Val289(Ile or Leu); Ser305Lys; Ser307Arg; Arg309Gln; andGln311Arg.
 9. The nucleic acid of claim 5, wherein the encodedpolypeptide comprises one or more substitution selected from the groupconsisting of: Lys27Glu; Asp29Asn; Asp40Asn; Met45Thr; Thr49Ala;Glu67Gly, His91Arg; Glu95(Ala or Thr), Val96Ala; Glu122Lys; Asn125Ala;Asn135Asp; Arg139His; Thr147Ala; Thr153Lys; Ser155Thr; Ser163Thr;Gln166(Arg or His), Ala172Thr; Ala173Val; Thr174Leu; Ala177Glu;Glu178Asp; Arg179Leu; Val180Gly; Ala201Ser; Val212Leu; Asp213Glu;Val215Ile; Ser226Arg; Lys244Arg; Gln251His; Val254Ile; Ser255Asn;Glu257Gly; Thr264(Ala or Ile); Thr272Met; Cys274Gly; Val275Ile;Lys280Arg; Ser281Asn; Lys285Asp; Lys286Arg; Phe290Ser; Thr291(Met orVal); Lys293Gln; Thr297Lys; Ile299(Thr or Val); Arg301His; Asn303Asp;Ser318Phe; Glu321Asp; Pro326Ser; Cys327Leu; and Ser328(Gly or Gln). 10.The nucleic acid of claim 5, wherein the encoded polypeptide has T-cellproliferative activity in the presence of a p35 polypeptide. 11-183.(canceled)